Biological measurement system

ABSTRACT

In a biological measurement system ( 10 ) for collecting one or more samples of sputum and/or mucus from a patient in the form of an aerosol and for analyzing said one or more samples to detect whether or not pathogens are present therein, the one or more samples are in solution within the system ( 10 ) and detection of the pathogens is performed using a fluorescently labeled assay. The system ( 10 ) is adapted to detect bacterial pathogens using evanescent-wave spectroscopy preferably by using a single-reflective technique. The one or more samples are advantageously provided to the system ( 10 ) in aerosol form. However, the system ( 10 ) is capable of being adapted for use in analyzing samples in liquid form. Methods of analyzing said one or more samples in the system ( 10 ) are described.

FIELD OF THE INVENTION

The present invention relates to biological measurement systems; moreparticularly, but not exclusively, the invention relates to a biologicalmeasurement system for providing early detection of respiratory disease,for example tuberculosis induced by the pathogen mycobacteriumtuberculosis.

REVIEW OF THE ART

Numerous biological measurement systems are known in the art fordetecting various forms of pathogens, for example bacteria, viruses,moulds and fungi.

In an article entitled ‘Detection of Antibody-Antigen Reactions at aglass-liquid interface as a Novel Optical Immunoassay Concept’,Proceedings of 2^(nd) Optical Fibre Conference (Stuttgart 1984) pp. 75,R. M. Sutherland et al. describe an optic waveguide apparatus wherein anantibody species is covalently immobilized onto a surface of a planar orfibre-optic waveguide. A sample solution comprising an antigen ispresented to the surface, whereat the antigen is immobilized by theantibody species. The antigen is interrogated using an evanescent wavecomponent of a light beam, totally internally reflected many timeswithin the wave-guide. The evanescent component exhibits acharacteristic that it penetrates only a fraction of its wavelength intoan aqueous phase at the surface whereat the antigen is immobilized;thus, the evanescent component is capable of optically interacting withsubstances, for example the immobilized antigen, bound to or very closeto the interface and only minimally with any bulk solution which mayinterface onto the surface.

Moreover, in a published paper entitled ‘Sensitivity enhancement ofevanescent wave immunoassay’ (1993) Yoshida et al. Meas. Sci. Technol. 4pp. 1077-9, there is described a fluoro-immunosensor suitable for thedetection of low concentrations of pathogens in blood and serum samples.The immunosensor employs an assay system including a sandwich assay in aflow cell. For a standard sandwich assay, a number of wash-steps arerequired. These wash-steps complicate the system and make it necessaryfor a relatively skilled operator to carry out the testing.

In a published United Kingdom patent no. GB 2174802, there is describedan optic-waveguide biosensor for detecting and monitoring specific assaymolecular species in flowing test fluid samples. The biosensor employs acomplex multiple reflection optical geometry wherein fluorescenceassociated with the binding of an antigen to an antibody-coated surfaceis characterized by an increase in the signal detected in the samedirection as that detected for the multiply reflected incident light. Adisadvantage of this sensor is that bulk scattering by an opticalwaveguide forming a part of the sensor can affect the signal leveldetected at the waveguide. Moreover, multilayer constructionconfigurations of the optical waveguide serve to further complicate thebiosensor and the complexity of signal levels observed. Again, theoperator of the biosensor must be relatively skilled.

In another published patent application no. GB 2227089, there isdescribed a system for the analysis of specific assay molecular speciesin test fluid samples. The system employs a detection method involvingthe use of detection of an evanescent wave component of an antibodyimmobilized on the surface of a planar or fibre optic waveguide. Thecoupling of the resonant wavelength is facilitated by an optical gratinglocated at either the interface between the dielectric body and themedium or between the dielectric body and the sensitized coating whichcan potentially result in alignment problems. Furthermore, the light isreflected many times within the waveguide and as such the intensityobserved is subject to losses due to scattering.

A European patent application no. EP 0519623 discloses an evanescentwave system comprising first and second wave propagating surfaces. Thefirst surface is used to detect the presence of a first analyte and thesecond is used to indicate the presence of a second analyte and/or areference. The system is complex and, in one embodiment, makes use ofthe inner and outer surfaces of the two wave propagating surfaces.

In a patent application no. EP 0239382, there is described another fibreoptic based device that has a high numerical aperture and does notutilize any cladding at its contact points. The device is of a complexdesign which employs a beam splitter and lens system susceptible toscattering losses. Again, the device incorporates a flow cell for theinterrogation of optically-detectable assays. This device is ofrelatively high cost and complexity.

An international PCT application no. PCT/US01/21634 concerns anapparatus and method for evanescent light fluoroassays, specificallyintended for use on bodily fluids. The apparatus is designed to detectmultiple spatially resolved assays to be read simultaneously usingdetection sensors such as a CCD camera, a photodetector, a photoarray orrelated sensors. Air pressure, vacuum or capillary action is used tomove the sample onto an assay area of a disposable cartridge. Again,this apparatus utilizes multiple reflections and, in one embodiment,these reflections occur in a very thin film, which improves measurementsensitivity. The apparatus relies upon personnel performing tests totransfer samples onto a disposable element of the apparatus and as suchis not a ‘safe’ method of handling pathogenic samples. Moreover, thepersonnel are required to be of a high skill level and the apparatustests for multiple conditions which is not the principle performancerequirement of the biological measurement system of the presentinvention.

A U.S. Pat. No. 5,922,550 relates to a sensitive device for thedetection of immunoassays. The basis of the device is somewhat differentto that of the present invention in that the sensitive device uses apredetermined pattern of analyte specific receptors that, in thepresence of the pathogen, produce a diffraction pattern from transmittedor reflected light. A diffraction image thereby generated can beobserved by eye or by an optical reading device.

A microassay rod and card system is described in a U.S. Pat. No.4,673,657. The rod and card system is intended for the simultaneousdetection of the presence of numerous different biologically importantsubstances in a single small sample. The device is rapid and makes useof standard immunoassay systems that are well documented in theliterature. The sample must be placed on the card system and so a safemeans of collection is not provided. Moreover, the detection of multiplepathogens is not desirable in the conditions for which the measurementsystem of the present invention is intended.

In a U.S. Pat. No. 3,992,516, there is described a direct fluorescentantibody composition and method for the detection of pneumocystiscarinii; the method of sample collection is not presented in the patent.

A German patent application no. DE 3932784 is concerned with a test foranalysing aerosols, including fluid from respiratory passages andspittle. Exhaled breath is collected directly into a mass spectrometeror is concentrated prior to analysis by collection onto a cooled plate.Analysis by mass spectrometry leads to the determination of themolecular species present in the gas/aerosol/liquid by fragmentation ofthese species and the subsequent determination of their masses.

Such an analysis results in a complex problem of co-addition todetermine all of the molecular species present. Collection on a cooledplate is a standard technique in the literature, namely matrixisolation, and has been utilized since the 1960's. The test relies ondirect measurement of the whole sample, rather than making use of achemical/biochemical assessment method The use of a mass spectrometer isexpensive and requires the incorporation of vacuum equipment; thus thistest is of high cost and is not portable.

In a United Kingdom patent application no. GB 2311856, an environmentalsampler is described for recovering particles having diameters in arange of 0.1 μm to 20 μm. In the sampler, a feed is used to coat thesurfaces of beads in a bead bed with liquid, which then entrapsparticles from an air sample. The liquid is then recovered and analysedfor the components that have dissolved in the liquid. Such an assaywould not be appropriate for the collection of pathogens containedwithin sputum/mucus from the upper lung area.

In an international PCT application no. PCT/AU95/00540, there isdescribed a nasal/oral filter designed for particle entrapment byinhalation or exhalation. The filter comprises a collection systemdesigned to fit into a user's mouth or nostril and has a non-linear pathto capture particulates. In this filter, the main target particles forcapture are potentially allergenic species, but the possibility existsto capture viruses or mycobacteria. The particles can be recovered bywashing or blowing through the sample collection system and subsequentlyanalyzed by culture, nucleic acid analysis or similar processes. Such anapproach means that the sample is transferred to another system whichimmediately gives rise to safety issues concerning safe sample handlingand speed of testing the samples.

An international PCT patent application no. PCT/SE96/00474 concerns adevice that investigates one or more components of exhaled air for thepresence of the pathogenic helicobacter pylori in the stomach andintestinal tracts of human beings. The device comprises a tubularelement for conducting exhaled air onto an airtight plate incorporatinga porous membrane for sample collection. Prior to producing the sample,the patient swallows an isotope-labeled, preferably radioactive, ureapreparation, which breaks down in the presence of helicobacter pylori.The preferred embodiment of the device indicates the presence ofradioactive carbon dioxide formed as a conversion product ofhelicobacter pylori. The plate absorbs the radioactive carbon dioxideand is subsequently removed from the device for radioactive analysis. Inthe detection of viruses and bacteria, such as mycobacteriumtuberculosis, the device is inappropriate as there is no simple wayprovided to isotopically label the pathogen nor is there any simple wayof forming a simple breakdown product.

In a U.S. Pat. No. 4,350,507, there is described a particle samplingapparatus for the collection of dust particles from the atmosphere. Theapparatus employs a grill and pre-filter system to remove largestnon-respirable particles and then a main filter to collect therespirable particles. This limits the dust concentration in someindustrial situations to tolerable limits. The apparatus is notdisclosed as being capable of performing biochemical assay analysis.

In a U.S. Pat. No. 5,372,126, there is described a pulmonary samplingchamber designed for the safe collection of pulmonary samples. Theapparatus entirely encloses the patient and, as such, is not portable.The primary aim of the system is to collect deep sample secretionsnon-invasively from a patient's lungs. Moreover, the chamber is equippedwith a replaceable exhaust filter unit to trap airborne pathogens andother harmful particles; the exhaust filter can subsequently be removedfor analysis and disposal.

A U.S. Pat. No. 3,745,991 describes a device for reducing environmentalcontamination during medical treatment and/or diagnosis. The aim of thedevice is to safely deliver and/or collect aerosol samples from apatient by enclosing the patient's face and passing any fluids generatedby exhalation from the patient through a filter system to collect anyhazardous materials for later disposal/analysis. The device is noteasily portable.

The Problem(s) to be Solved Accordingly

It is becoming increasingly important for organisations, for examplegovernment agencies and humanitarian relief agencies, to have at theirdisposal facilities for the rapid detection and identification ofpathogens. Such pathogens include newly emerging viruses andre-emergence of known diseases such as bubonic plague, tuberculosis andcholera. On account of such pathogens becoming increasingly resilient tomedication, there is a considerable need for measurement systems thatcan be used for the early detection of pathogen outbreaks so thatisolation measures and targeted medication can be applied to containfurther pathogen spread.

Moreover, on account of outbreaks of disease often occurring ineconomically less advanced regions of the world and being spread byvectors such as aviation to other regions thereof, there is a need formeasurement systems which are relatively inexpensive, which arestraightforward to use by untrained staff, which can give results at thepoint-of-test/care, and which are potentially less susceptible toinadvertently spreading pathogens when operated by untrained staff.

In particular, the detection of bacterial infection is of vitalimportance in global terms. Field-testing of bacterial infection,preferably using a test that responds rapidly, is particularly desirablebecause of the prevalence, virulence and major impact of majorinfections such as pneumonia, tuberculosis, malaria and other pathogens.Contemporary bacterial tests are mainly based on complex laboratoryassays and are therefore potentially expensive and are not especiallysuitable for field use. Moreover, many contemporary tests require asubstantial time period, for example in a range of 2 to 4 weeks, toprovide a positive identification of the presence of pathogens. Morerecently, rapid tests have been developed which offer reducedidentification timescales to hours/days. These rapid tests are primarilybased on the analysis of sputum/mucus samples from upper lung regions;however, the collection and handling of such samples is hazardous topersonnel conducting and/or supervising such testing. Thus, bothtimescale and potential pathogen transmission problems make thesecontemporary tests difficult to execute in field locations.

Furthermore, at the current state of the art for the field-testing oftuberculosis (TB), a ‘standard’ skin test employed is compromised by HIVstatus and so the only method currently used in the third world is thatof smear microscopy on sputum/mucus samples. The accuracy of these testsis dependent on skilled operators and frequent re-tests.

In general, in these circumstances, a significantly safer method ofpulmonary bacterial sensing is required for addressing the pathogentransmission problems that are prevalent when collecting and handlingsamples for subsequent pathological analysis.

Furthermore, rapid and reliable detection of other types of medicalcondition, for example hormonal abnormalities in the context of steroid(hormone) abuse in professional sport, markers for cancer and so on isalso highly desirable.

None of the known systems reviewed above adequately addresses theseproblems.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is providedbiological measurement system for measuring the concentration ofcomponents included in a sample, the system characterised in that itcomprises:

-   (a) collecting means for collecting the sample;-   (b) concentrating means for spatially concentrating the sample;-   (c) marking means for optically labeling the components present in    the concentrated sample; and-   (d) interrogating means for optically interrogating the labeled    components and thereby generating a measure of the concentration of    components present in the sample.

Preferably, the collecting means is adapted for collecting the sample inaerosol form. Aerosol sample collection is of benefit where the systemis employed for testing pulmonary diseases and environmental air-bornepollutants. It maximizes the pathogen collectable by comparison withconventional (sputum-only or mucus-only) “cough”-style methods.

Preferably, the collecting means further comprises nebulizing means foremitting a mist for inducing aerosol emission from a subject. Thenebulizing means is of benefit in that is capable of increasing theamount of sample obtained when performing pulmonary testing.

More preferably, the nebulizing means in use is adapted to generate asaline mist comprising saline droplets having diameters in a range of 6μm to 20 μm. The range is of advantage in that droplet sizes of lessthan 6 μm is too palatable to induce coughing whereas droplet sizesgreater than 20 μm can be unpleasant to inhale.

Most preferably, the saline droplets have diameters in a range of 10 μmto 15 μm and comprise saline solution having a saline concentration in arange of 0.1% to 2% by weight.

Preferably, the concentrating means further comprises a feature forscraping surfaces where the sample is deposited to spatially concentratethe sample. Spatial concentration of the sample is capable of enhancingthe measurement sensitivity of the system.

Preferably, the feature is elastically deformable for spreading thespatially concentrated sample over an optical interrogation regionwhereat the concentrated sample is subjected to optical interrogation.

Beneficially, the marking means comprises at least one of a selectivebinding assay and a competitive displacement assay for optically markingpresence of the components by way of fluorescent markers. Such assaysare capable of providing effective interface between biochemical andoptical domains.

Preferably, the fluorescent markers are bound to antibodies for use inat least one of the selective assay and the competitive assay.Antobodies, for example as employed in immunoassay, are of advantage inthat they can be made highly selective with remain to moleculargroupings or microbes to which they are capable of binding. Moreover,antibodies are presently becoming relative cheap to manufacture in bulkusing contemporary genetic engineering processes.

Preferably, the fluorescent markers are fluorophores bound to theantibodies by way of an intermediate carrier such that a plurality offluorophores are associated with each antibody. The use of fluorophoresis of advantage in that they are capable of being excited by opticalradiation at a first radiation frequency and emitting fluorescentradiation at a second radiation frequency, the first and secondfrequencies being mutually different, thereby enabling the excitationradiation and the emitted fluorescent radiation to be individualisolated.

More preferably, the intermediate carrier is implemented in the form oflatex spheres.

The interrogating means beneficially comprises an optical evanescentdetector for detecting changes in optical response induced by thepresence of the components. Evanescent wave interrogation is especiallyadvantageous as it enables a planar optical surface across which thesample is spread to be specifically targeted for interrogation.

Preferably, the evanescent detector includes:

-   (a) one or more of a diode laser and a LED as a source of    interrogating radiation for interrogating the concentrated sample;    and-   (b) one or more of a avalanche photodiode, a photodiode array and a    photomultiplier tube as an optical detector for detecting    fluorescent radiation emitted from the concentrated sample in    response to optical interrogation of the sample, the optical    detector for generating a detection signal indicative of changes in    fluorescence from the sample resulting from the presence of the    components in the sample.

Such sources and detectors of optical radiation are of advantage in thatthey are potentially inexpensive, compact and robust.

Preferably, the system further comprising strobing means for strobingradiation emitted from the source of interrogating radiation, andsynchronous demodulating means for demodulating the detection signal insynchronism with the strobe to render the system less sensitive toquasi-constant optical radiation received at the optical detector. Suchstrobing is capable of rendering the system less influenced by theeffects of stray ambient illumination penetrating into the system.Moreover, such a strobe also enables effects of offset voltages withinelectronic components of the detecting means on the measurement to besignificantly reduced.

Preferably, the system further comprising computing means for changes inthe detection signal when the components in the sample are opticallylabeled or displace optical labels.

More preferably, the computing means is arranged to monitor theconcentrated sample before and after fluorescent labeling thereof tocalculate the measure of the concentration of the components in thesample. Such dual measurement is of benefit in removing effects ofsystematic errors in the system, for example background fluorescenceoccurring in the interrogating means.

Beneficially, the computing means further comprises one or more of:

-   (a) displaying means for displaying the measure of the concentration    of the components in the sample, and-   (b) data logging means for storing a record of measure of the    concentration of the components.

Preferably, the collecting means is arranged to enclose the sample,thereby preventing personnel contact with the sample when the system isin use. Such containment is of advantage in assisting to prevent thespread of dangerous pathogens and also renders the system safer in use.

More preferably, the collection means is arranged to be a single-usedisposable part. Such single-use is of further advantage in preventingthe spread of potentially dangerous pathogens. Most preferably, thecollecting means comprises features rendering it substantiallyundismantleable after sample collection therein.

Preferably, the collecting means comprises vortex enhancing means fordeposition of the sample within the collecting means.

The collecting means preferably comprising filtering means for at leastpartially inhibiting spread of the components of the sample from thecollecting means.

Preferably, the marking means includes lysing means for causing lysis ofthe components present in the sample, thereby enhancing measurementsensitivity of the system by increasing the number of availablepotential optical labeling sites.

The system according to the first aspect is capable of being used in awide range of applications not limited to the biological domain. Inparticular, but not exclusively, the system is preferably adapted toidentify the components in the form of one or more of the following:

-   (a) antibodies;-   (b) nucleic acids;-   (c) enzymes and/or other proteins;-   (d) analogues of one or more of (a) to (c); and-   (e) a microorganism.

With regard to microorganisms, the system is especially appropriate forthe detection of one or more of the following:

-   (a) a virus;-   (b) spores;-   (c) molds;-   (d) pollen; and-   (e) a microbiological allergen.

Moreover, the system is also preferably adapted to identify thecomponents in the form of one or more of the following:

-   (a) toxic dust;-   (b) an explosive;-   (c) a drug; and-   (d) a pollutant.

According to a second aspect of the present invention, there is provideda method of detecting one or more pathogens in one or more samples ofsputum from a subject using a system according to the first aspect ofthe invention, the method involving the steps of:

-   (a) collecting said one or more samples in the collecting means;-   (b) spatially concentrating the one or more samples in the    concentrating means;-   (c) optically labeling one or more pathogens present in said one or    more samples;-   (d) optically interrogating the pathogens to achieve an optical    response; and determining from the optical response of said one or    more samples whether or not said one or more pathogens are present    in said one or more samples.

Preferably, in steps (b) and (c), a fluorescently labeled assay isemployed to provide the optical response.

Preferably, in steps (b), (c) and (d), detection of fluorescence isperformed using evanescent-wave spectroscopy.

Preferably, when executing the method, said one or more pathogenscomprise one or more of:

-   (1) antibodies;-   (2) nucleic acids;-   (3) enzymes or other proteins;-   (4) analogies of (1) to (3); and-   (5) a micro-organism.

The method is advantageously adapted for the detection of bacteriaassociated with pulmonary and pulmonary-related infections.

Moreover, the method is preferably adapted for the detection of one ormore of the following pathogens:

-   (1) a virus;-   (2) a protein and/or antibody;-   (3) another symptomatic particle not included in (1) or (2);-   (4) a spore;-   (5) a mold;-   (6) pollen;-   (7) an allergen;-   (8) toxic dust;-   (9) an explosive;-   (10) a drug; and-   (11) a pollutant.

Preferably, to enhance aerosol generation, the inhalation of one or moreof:

-   -   esters, water vapour, saline vapour, expectorant and menthol is        used to assist release of bacteria-containing mucus from the        trachea or from the upper lung of a subject being tested.

Beneficially, a partial negative pressure is employed to assist inobtaining said one or more samples in aerosol form.

The method is capable of being applied to testing a diverse range ofsamples. For example, said one or more samples preferably comprise anaerosol of blood or other bodily fluid or bodily fluid in liquid form.

In the method, analysis of said one or more samples is performed usingone or more of:

-   (a) an ELISA chromogenic reaction; and-   (b) a surface acoustic wave (SAW) biosensor to detect an antigen in    said one or more samples.

According to a third aspect of the present invention, there is provideda sample collection apparatus or collecting aerosol samples,characterised in that the apparatus comprises:

-   (a) collecting means for collecting the sample; and-   (b) concentrating means for spatially concentrating the sample.

Preferably, the collecting means further comprises nebulizing means foremitting a mist for inducing aerosol emission from a subject.

Preferably, the nebulizing means in use is adapted to generate a salinemist comprising saline droplets having diameters in a range of 6 μm to20 μm.

More preferably, the saline droplets have diameters in a range of 10 μmto 15 μm and comprise saline solution having a saline concentration in arange of 0.1% to 2% by weight.

Beneficially, the concentrating means further comprises a feature forscraping surfaces where the sample is deposited to spatially concentratethe sample.

Preferably, the feature is elastically deformable for spreading thespatially concentrated sample over an optical interrogation regionwhereat the concentrated sample is subjected to optical interrogation.

Preferably, the apparatus further comprises an interrogation regionwhereat the sample in spatially concentrated, the interrogation regionbeing susceptible to optical interrogation.

Preferably, the interrogation region is susceptible to evanescent waveinterrogation.

Preferably, the collecting means is arranged to enclose the sample,thereby preventing personnel contact with the sample when the apparatusis in use.

To reduce the potential spread of dangerous pathogens, the collectingmeans preferable comprises features rendering it substantiallyundismantleable after sample collection therein.

Preferably, the collecting means comprises vortex enhancing means fordeposition of the sample within the collecting means. Vortex enhancingmeans include one or more of a septum and a bend in a sample collectionregion.

To reduce the risk of spreading potentially dangerous pathogens, thecollecting means preferably comprises filtering means for at leastpartially inhibiting spread of the components of the sample from thecollecting means

According to a fourth aspect of the present invention, there is providedan immunosensor for collecting one or more samples of sputum from apatient in the form of an aerosol and for analysing said one or moresamples to detect whether or not pathogens are present therein.

The immunosensor is capable of providing a significantly safer method ofpulmonary testing, the sensor being designed for safe handling of testsamples.

Preferably, in the immunosensor, said one or more samples are insolution within the sensor and detection of the pathogens within saidone or more samples is performed using a fluorescently labeled assay.The fluorescently labeled assay is capable of providing a sensitive andreliable approach to detecting presence of the pathogens.

More preferably, the detection of bacterial pathogens is performed usingevanescent-wave spectroscopy or fluorimetry. The use of evanescent-wavespectroscopy or fluorimetry enables optical interrogation to be appliedefficiently to a relative small sample of pathogen to detect itspresence.

Preferably, the immunosensor is adapted to detect one or more of thefollowing pathogens:

-   (a) antibodies;-   (b) nucleic acids;-   (c) enzymes and/or other proteins;-   (d) analogues of one or more (a) to (c); and/or-   (e) a micro-organism.

More preferably, the immunosensor is adapted for the detection ofbacteria in a sample, the bacteria being associated with pulmonary andpulmonary-related infections. Alternatively, or additionally, theimmunosensor is adapted for the detection of one or more of:

-   (a) a virus;-   (b) a protein and/or antibody;-   (c) other symptomatic particles not included in (a) and (b); for    example indicators of forms of cancer.-   (d) spores;-   (e) molds;-   (f) pollen;-   (g) an allergen;-   (h) toxic dust;-   (i) an explosive;-   (j) a drug; and-   (k) a pollutant.

According to a fifth aspect of the present invention, there is provideda method of detecting one or more pathogens in one or more samples ofsputum from a patient using an immunosensor according to the fourthaspect of the invention, the method involving the steps of:

-   (a) collecting said one or more samples in the immunosensor;-   (b) fluorescently labeling one or more pathogens present in said one    or more samples;-   (c) interrogating said one or more samples using optical    interrogation to achieve an optical response; and-   (d) determining from the optical response of said one or more    samples whether or not said one or more pathogens are present in    said one or more samples.

Preferably, in steps (b) and (c), a fluorescently labeled assay isemployed to provide the optical response.

More preferably, for efficiently interrogating a relatively smallquantity of sample, detection of fluorescence in steps (b), (c) and (d)is performed using evanescent-wave spectroscopy or evanescent-wavefluorimetry.

Preferably, the method is susceptible to detecting the occurrence ofsaid one or more pathogens by way of:

-   (1) antibodies;-   (2) nucleic acids;-   (3) enzymes or other proteins;-   (4) analogies of (1) to (3); and-   (5) a microorganism.

More preferably, the method is adapted for the detection of bacteriaassociated with pulmonary and pulmonary-related infections.

Alternatively, or additionally, the method is preferably adapted for thedetection of one or more of the following pathogens:

-   (1) a virus;-   (2) a protein and/or antibody;-   (3) another symptomatic particle not included in (1) or (2);-   (4) a spore;-   (5) a mold;-   (6) pollen;-   (7) an allergen;-   (8) toxic dust;-   (9) an explosive;-   (10) a drug; and-   (11) a pollutant.

In order to induce more efficient sample generation, the methodpreferably involves the inhalation of one or more of:

-   -   esters, water vapour, saline vapour, expectorant and menthol to        assist release of bacteria-containing mucus from the trachea or        from the upper lung of the patient. The patient, in this case,        may be either a human being or an animal.

Inhalation of the vapour should be from a separate vessel such as, butnot exclusively, a simple nebuliser, from within the sample collectionsystem or via an inlet tube to the sample collection system. Inhalationmay take place via a pipe which may or may not incorporate a demandvalve, diaphragm valve or similar.

Exhalation may be via a ‘plug’ in the pipe that ruptures to allow‘breath’ to enter the chamber and activate the fluorophore markedantibody.

The sample is collected directly onto a prism in the sample collectionsystem.

Exhalation by the patient is via a large inlet pipe. The aerosol exitsthe sample collection system via a smaller diameter pipe, into a filteror sample collection bag. The effect of the two different diameters isthe creation of a ‘swirl’ effect in the vessel.

Exhalation into the sample collection vessel may be via a plug thatruptures to release a hydrating agent, such as PBS or water, and/or thedesired antibodies and/or fluorescent markers into the sample collectionvessel. Further, exhalation into the sample collection vessel may be viaa pipe or pipe containing a venturi. The exit pipe of the samplecollector may also incorporate a venturi.

A partial negative pressure may be employed to assist in obtaining saidone or more samples in aerosol form.

Preferably, said one or more samples comprise an aerosol of blood orother bodily fluid, such as urine, pathogenic sera, semen, saliva, tearsor sweat. The analysis of these fluids significantly increases the rangeof pathogens that can be tested. Tests on saliva can, for example, becarried out to detect streptococcus and staphylococcus.

The method is preferably adapted to analyse one or more samples ofnon-biological origin.

The interrogation technique is also adapted for use to detect all of thepathogens described above, with liquid samples of bodily fluids such asblood, urine, pathogenic sera, semen, saliva, tears or sweat and othersamples such as food and non-biological samples.

The samples, either in the form of aerosol or liquid, may be dilutedusing PBS, water or other appropriate solvent.

The method is preferably adapted to cope with particles ofnon-biological origin including at least one of an environmental aerosoland an effluent.

More preferably, analysis of said one or more samples is performed usingone or more of:

-   (a) an ELISA chromogenic reaction; and-   (b) a surface acoustic wave (SAW) biosensor to detect an antigen in    said one or more samples.

Preferably, the sensor is used to execute a test which analyses exhaledbreath, which is in the form of an aerosol, comprising bacteria or otherpathogens to be detected contained in water or sputum droplets of suchbreath. The aforesaid one or more samples are preferably collecteddirectly into a sample tube for testing using, for example, afluorometric assay.

Hydration, using PBS, water or other suitable solvent may be required inorder to differentiate between bulk and surface fluorophores.

A fluorometric assay technique may require time for culture to increasethe sample numbers to aid detection.

Preferably the sample collection system will be shatterable orsplinterable after use for safe disposal after a single use.

Fluorimetry has been shown to be of considerable importance for thedetection of biological materials such as proteins and DNA, wherefluorophores on antibodies are used as markers for detection. Detectionusing such fluorimetry can be executed by way of either

-   (a) bulk fluorescence measurements; or-   (b) through the application of interrogation techniques such as    evanescent wave detection; or-   (c) cavity ring down spectroscopy; or-   (d) through the use of displacement assays.

Such fluorimetry offers some potential advantages in terms ofspecificity, simplicity, and sensitive. Evanescent wave detection iswell known, but low-cost evanescent wave fluorimeters are not yetcommercially available for use in pathogen detection as described withrespect to the present invention.

The inventors are unaware of prior art regarding developments on thedetection of pulmonary bacterial infectious agents from exhaled breathusing fluorimetry. The present invention represents an improvement overexiting techniques because it reduces the level of expertise required tocarry out an assay test; moreover, hazard associated with potentialtransmission of diseases to the tester from handling contaminatedsamples is reduced. The inventors have therefore devised an immunosensorwhich is fast acting, low cost, and portable with disposable sampleholders; the immunosensor is especially susceptible to use in fieldenvironments, for example in third-world countries. It is designed forscreening large patient numbers and retesting as appropriate in forexample, schools and institutions.

In the sixth aspect present invention, there is provided a samplecollection apparatus comprising:

-   (a) a sample collection volume bounded by an interior surface for    receiving a gaseously-borne sample;-   (b) A sample collection volume bounded by an interior surface for    receiving a liquid sample where the liquid is sprayed into or added    dropwise into the sample collector, and-   (c) collecting means for collecting, in use, at least a portion of    the sample deposited on the interior surface and for concentrating    the portion at a test location susceptible to subsequent    interrogation.

The invention is of advantage in that the apparatus is capable ofeffectively and conveniently collecting the sample for analysis.

Preferably, the collection volume is provided with vortex generatingmeans for causing, in use, an incoming jet transporting thegaseously-borne sample to form into one or more vortices to assist withdeposition of the sample onto said interior surface.

Vortex flow in a fluid carrying a particulate load results in conversionof kinetic energy in the flow to thermal dissipation therein and asubsequent deceleration with a resulting deposition of the particulateload transported within the flow.

Preferably, the collection volume is implemented as a tubular elementand the collecting means is implemented as a plunger element arranged toslidably engage within the interior surface of the tubular element. Suchan arrangement is of advantage in that the tubular element is convenientfor offering to users' mouths and for hand-held support, whereas theplunger element is capable of sealing an end of the tubular element and,when pushed into the tubular element, assisting to spatially concentratethe sample.

More preferably, the plunger element forms a sufficient seal onto thetubular element for collecting the sample into a ring-like mass when theplunger element, in use, is slidably moved within the tubular element.

Preferably, the plunger element includes an end region comprising aprojection susceptible to collecting the ring-like mass together whenthe plunger element is rotated relative to the tubular element. Theprojection is capable of functioning in a spoon-like manner to scoop upthe sample from the tubular element to concentrate it into one spatiallocation.

The plunger element advantageously includes at its end region, opticalinterfacing means for interfacing between optical interrogating meansand the sample, thereby enabling the optical interrogating means tointerrogate the sample via the optical interfacing means. Use of opticalinterrogating means is of benefit in that non-contact interrogation ofthe sample can be achieved, thereby, in the case of contagiouspathogens, reducing the risk of spreading disease further.

Preferably, the plunger element comprises a hollow interior region forreceiving, in use, the optical interrogating means. Concentric mountingof the interrogating means within the plunger element, and concentricmounting of the plunger element within the tubular element is of benefitin enabling the interrogating means to be brought in close proximity,for example within a few mm, of the sample. Moreover, such concentricmounting also renders the apparatus potentially highly compact.

Preferably, the tubular element and the plunger element are designed tobe disposable items whereas the optical interrogating means is designedto be a non-disposable item. Such disposability is of advantage when thetubular element and the plunger element are used to collect samplesincluding pathogens that are potentially contagious; the tubular elementand the plunger element can, for example, be disposed of by incinerationto circumvent spread of undesirable pathogens. More preferably, thetubular element and the plunger element are designed to be mutuallyinterlocking after sample collection has occurred therein to preventthese elements being reused with associated risk of cross-contamination.

The optical interfacing means preferably comprises a prism for guidinginterrogating radiation from the interrogating means to the sample, andfor guiding response radiation from the sample back to the interrogatingmeans. Use of a single optical component for bi-directional opticalradiation propagation enables the cost and size of the apparatus to bepotentially reduced. More preferably, the prism is a dove-type prism;such a prism is susceptible to being used, for example, inevanescent-wave optical interrogation of samples, especially samplessubjected to fluorophore tagging.

The interrogating means comprises a source of strobed radiation forproviding the interrogating radiation, and a photodetector andassociated demodulator for detecting response radiation from the sampleand for demodulating the response radiation with respect to the strobe.Such a strobe arrangement can be applied to discriminate ambientquasi-constant optical radiation contributions, for example as a resultof light leakage into the apparatus from its ambient environment.

Preferably, for ease and cheapness of manufacture, one or more of thetubular element and the plunger element are fabricated from plasticsmaterials. More preferably, the plastics materials comprise one or moreof an acrylate, polyethylene, polypropylene, silicone rubber, polyvinylchloride (PVC), alkylene, polycarbonate, and polytetrafluoroethylene(PTFE) plastics material. Most preferably, the plastics materials areinjection moulded.

According to a seventh aspect of the invention, there is provided amethod of collecting a sample from a user utilizing an apparatusaccording to the first aspect of the sample collection system, themethod comprising the steps of:

-   (a) exhaling mucus droplet borne air from the user into a collection    volume of the apparatus;-   (b) depositing mucus droplets from the exhaled air onto an interior    surface of the collection volume;-   (c) collecting the droplets together from the surface using    collecting means of the apparatus to provide a collected mass of    droplets.

Preferably, the method further comprising the step of interrogating thecollected mass of droplets after step (c) to determine one or morecharacteristics thereof. More preferably the collected mass isinterrogated optically.

Preferably, in the method, the collected mass is arranged to fluorescein response to being optically interrogated, and the one or morecharacteristics determined from the fluorescence.

Preferably, in step (b) of the method, the exhaled air is arranged toflow in vortices to promote deposition of the droplets onto the interiorsurface.

It will be appreciated that features of the invention described in theforegoing can be combined in any workable combination falling within thescope of the invention as defined by the final claims.

DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the following diagrams in which:

FIG. 1 is a schematic diagram of a biological measurement systemaccording to the invention;

FIG. 2 is a schematic illustration of operation of a sample collectionunit of the measurement system of FIG. 1;

FIG. 3 is an illustration of a sample collection tube of the measurementsystem of FIG. 1;

FIGS. 4 a to 4 c are illustrations of a plunger suitable for use withthe collection tube of FIG. 3;

FIG. 5 is an illustration of an electronics module of a reader unit ofthe measurement system of FIG. 1;

FIG. 6 is an illustration of alternative sample collection tubes for themeasurement system of FIG. 1;

FIG. 7 is a schematic diagram of a yet further alternative samplecollection tube for the measurement system of FIG. 1;

FIGS. 8 a and 8 b are illustrations of optical components includedwithin the electronics module of FIG. 5;

FIG. 9 is a schematic diagram of a sealing cap included within thesample collection unit of FIG. 2;

FIG. 10 is a schematic representation of an optical configurationemployed within the measurement system of FIG. 1;

FIG. 11 is an illustration of a modification to the measurement systemof FIG. 1 for the analysis of liquid samples such as blood;

FIG. 12 is a schematic diagram of a compact dove prism for incorporationinto the measurement system of FIG. 1;

FIGS. 13 and 14 are depictions of a selective binding assay employed inthe system of FIG. 1; and

FIG. 15 is a depiction of a competitive displacement assay employed inthe system of FIG. 1.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In the following description, embodiments of a biological measurementsystem will initially be described in overview. Later, component partsof the embodiments and their associated biochemistry will be describedin more detail.

The system described herein employs evanescent wave spectroscopy andevanescent wave fluorimetry to detect the presence of a pathogenicsubstance using an immunoassay technique.

1. System Overview

Referring firstly to FIG. 1, there is shown a biological measurementsystem according to the invention. The system is indicated generally by10 and comprises a sample collection unit indicated by 30, and acorresponding complementary reader unit indicated by 50. For displayingtest results, the reader unit 50 includes a readout display 60. Thecollection unit 30 is adapted for collecting exhaled material from auser 40, such material providing test samples for subsequent analysis.

The collection unit 30 is designed to engage mechanically into thereader unit 50. Moreover, the collection unit 30 is sufficiently compactfor it to be hand-held by the user 40. Furthermore, the collection unit30 is implemented in the form of a hollow sample tube 70 comprising:

-   (a) an input orifice 80 for engaging onto a mouth region of the user    40;-   (b) an intermediate orifice 90 for coupling to a gas collecting    region, for example to an inflatable bag 100; and-   (c) an access orifice for a piston-like plunger 110 which is    slidably and rotationally moveable within the sample tube 70.

In order to reduce a risk of cross-contamination from one user toanother, the collection unit 30 is designed to be a disposable item;namely, the collection unit 30 is used only once to collect the samplefor testing and to safely present the sample to the reader unit 50 forinterrogation. The collection unit 30 is preferably molded from aplastics material to render it relatively inexpensive to manufacture,and also to render it susceptible to incineration to reduce the spreadof potentially dangerous pathogens collected therein. Moreover, thecollection unit 30 is designed so that the reader unit 50 is preventedfrom coming into direct contact with collected samples within the tube70 which can potentially comprise dangerous pathogens.

2. Overview of System Operation

Operation of the biological measurement system 10 will now be describedin overview with reference to FIGS. 1 and 2.

After manufacture including the deposition of active biomaterials, thecollection unit 30 is preferably sealed within a desiccatedhermetically-sealed package for storage prior to deployment. Such apackage potentially prevents moisture from denaturing the aforementionedactive biomaterials and also potentially reduces the risk of thecollection unit 30 unintentionally becoming contaminated with pathogensprior to use; thus, such a package assists to prevent the system 10 fromyielding unrepresentative test results.

Step 1: Immediately prior to deployment, the user 40, or a personsupervising testing, removes the collection unit 30 from its hermeticpackage. The user 40 then engages his/her mouth to the input orifice 80of the sample tube 70.

Step 2: Next, if required to assist in the production of a sample fromthe user 40, a saline mist is generated either from within the sampletube 70, for example from a miniature pressurized gas canister atomizercoupled thereto, or within a nebulising device remotely connected to thecollection unit 30; conveniently, the nebulising device is afoot-operated pump-like device. The user 40 inhales the saline mist viathe input orifice 80, the mist inducing sufficiently vigorous coughingfor the user 40 to exhale sputum and/or mucus in the form of an aerosolthrough the orifice 80 into the sample tube 70. The aerosol passes intothe tube 70 and is encouraged by the aerodynamic internal profile of thetube 70 to circulate and decelerate in a vortex-like trajectory todeposit mucus and/or sputum onto internal walls of the tube 70.Preferably, a collection volume, for example the inflatable bag 100 ofplastics material such as polyethylene or polyvinyl chloride (PVC),connected to the intermediate orifice 90 is included to receive airexhaled by the user 40; each cough can amount to two liters volume ofair, hence the bag 100 is conveniently sized to accommodate severalcoughs. More preferably, the collection bag 100 is provided with an gasexit orifice 105 comprising a fine filter having a pore size which issufficiently large to enable the bag 100 to deflate over a period ofseveral tens of seconds, thereby rendering the bag 100 subsequentlyconvenient in size to handle when deflated, but also sufficiently smallto substantially prevent spread of potential pathogens exhaled by theuser 40. Moreover, the intermediate orifice 90 is of moderate flowresistance relative to the input orifice 80 and exit orifice 105 and ispreferably included between the sample tube 70 and its associated bag100 so as to enhance the aforesaid vortex gas trajectory and promoteefficient deposition of mucus and/or sputum within the sample tube 70.

Alternatively, a sample of saliva can be collected from the test subjectvia the action of spitting into the sample collection apparatus.

Step 3: When a sufficiently large sample of sputum and/or mucus iscollected within the collection unit 30, a sealing cap (not shown inFIG. 1, but denoted by 900 in FIG. 2) is placed over the input orifice80. Next, the plunger 110 is actuated to mechanically concentrate thesputum and/or mucus within an interrogation region, for example anoptical surface 120 of the plunger 110; in particular, the sputum and/ormucus is concentrated onto the optical interrogation surface 120provided at an end face of the plunger 110, the optical surface 120being capable of supporting evanescent interrogation-radiationpropagation which will be described in more detail later. Preferably,the plunger is both pushed and rotated within the sample tube 70 tomechanically concentrate the test sample at the surface 120. Morepreferably, the plunger 230 is rotated by at least 360° to ensure thatas much of the sample as possible is collected onto a sample collectionprojection of the plunger.

An incubation period may be needed prior to optically interrogating thesample to generate a measurement reading.

Step 4: When the plunger 110 has been pushed substantially fully intothe sample tube 70 to fully collect the sample onto the optical surface120, the collection unit 30 is then offered to the reader unit 50 sothat a projection 130 thereof couples into the plunger 110 to enableoptical interrogation of the optical surface 120 for determining opticalproperties of the sample thereat; such optical interrogation ispreferably achieved by way of evanescent light propagation at thesurface 120. Results of the optical interrogation are presented on thedisplay 60 to the user 40 and/or associated tester to establish whetheror not the user 40 is infected with one or more pathogens, for examplemycobacterium tuberculosis, to which the system 10 is responsive.

This completes an overview of operation of the system 10.

One or more of the sealing cap, the collection tube 70 and its plunger110 can have incorporated therein one or more reservoirs of liquid fortreating the optical surface 120 prior to optical interrogation thereof.Such puncturable reservoirs preferably contain buffer solutions orreagents such as, but not exclusively:

-   (a) lysing agents for causing collected pathogens, for example    mycobacterium, to fragment;-   (b) rinsing agents for rinsing displaced fluorophores from the    optical surface 120 and/or flooding the optical surface 120 in    fluorophores coupled to pathogen-selective antibodies;-   (c) thinning agents to break up mucus; and-   (d) developing agents for the sample such as labeled antibodies.

Reagents may be in the form of solids such as a lyophilized sphere toprotect them during storage; such solid reagents may be present in oneor more reservoirs or in the sample collection tube 70.

These one or more reservoirs are preferably arranged to be userpuncturable to deliver their contents after sample collection but priorto optical interrogation. Mechanical construction of the reservoirs willbe described later with reference to FIG. 9.

As will be further described later, the optical surface 120 is oneoptical face of a prism configured to support evanescent light radiationpropagation therealong. The prism is preferably implemented as a doveprism, although alternative types of prism can be employed.

3. System Component Parts

Detailed design of individual components of the system 10 will now bedescribed.

3.1 Sample Collection Unit

Referring next to FIG. 3, there is shown the hollow sample tube 70implemented as a substantially cylindrical hollow sample tube 200. Thetube 200 comprises a first open end indicated by 210 for receiving anexhaled sample from the user 40; the first open end 210 corresponds tothe input orifice 80 in FIG. 1. Moreover, the tube 200 further comprisesa second end 220 for receiving a hollow plunger 230; the plunger 230corresponds to the plunger 110 of FIG. 1. The tube 200 also includes asubstantially cylindrical side tube 240 serving as the intermediateorifice 90, the side tube 240 having an associated longitudinal centralaxis substantially orthogonal to that of the sample tube 200. At aregion where the tubes 200, 240 adjoin, there is preferably included amesh or filter gauze 250. The tube 200 further comprises a peripheralring 260 around the first end 210 so that this end 210 is substantiallydevoid of any sharp edges which could injure the user 40 manipulatingthe tube 200, for example when the user 40 manipulates the tube 200towards his/her mouth.

The hollow plunger 230 is also of substantially cylindrical form andfabricated to be slidably and rotationally moveable concentricallywithin the inside of the sample tube 200 as illustrated, the tube 200and the plunger 230 being a mutually precise fit. Preferably, theplunger 230 is provided with a resiliently deformable sealing ring (notshown) substantially at an end of the plunger 230 offered to the sampletube 200 when in use. The sealing ring is preferably fabricated from anitrile rubber material, for example proprietary Viton material,silicone or polytetrafluoroethylene (PTFE). Moreover, the sealing ringis advantageously devoid of any lubricating material, for examplesilicone grease, which could potentially contaminate samples collectedwithin the tube 200, and thereby compromise system 10 operation.Additionally, vapour emitted from a lubricant may potentially be harmfulto the user 40 if ingested.

The hollow tube 200 is additionally provided with a saline atomizingassembly indicated generally by 300 at a region of the tube 200 near tothe first open end 210. The assembly 300 is preferably coupled to anebulizer, for example a foot-pump operated device, for forcing salinesolution at pressure to the assembly 300 for generating a divergent jet310 of saline mist for inhalation by the user 40; the saline mist iseffective at promoting vigorous coughing to induce user 40 ejection ofsputum and/or mucus. Preferably, the jet 310 comprises saline dropletshaving a diameter in a range of 6 μm to 20 μm. More preferably, thesaline droplets have a diameter in a range of substantially 10 μm to 15μm. A saline solution from which the droplets are generated preferablyis of a concentration in a range of 0.1% to 2% by weight of sodiumchloride to water; more preferably, the saline solution is of aconcentration in a range of 0.7% to 1.1% by weight. The assembly 300includes a substantially central capillary tube 320 which is angled atits nozzle end towards the first open end 210 to reduce an amount ofsaline mist swept towards the plunger 230. Preferably, the assembly 300is recessed relative to the inside bore of the hollow tube 200 so thatthe plunger 230 can be advanced towards the first end 210 beyond aregion where the assembly 300 is connected to the hollow tube 200 asillustrated. The assembly 300 is preferably integrally molded as part ofthe hollow tube 200; alternatively, in order to simplify molding toolsrequired, the assembly 300 can be a snap-fit retained insert which isassembled into a projecting side port of the hollow tube 200 duringmanufacture. If required, the side port can be molded with its centralaxis orientated towards the first end 210 so that the insert does notrequire its capillary tube 320 to be shaped towards this end 210.

Induction of more efficient sample generation may alternatively beaccomplished by the inhalation of one or more of water vapour, esters,expectorant and/or menthol.

In operation, the plunger 230 is retracted so that its end surfaceindicated by 270 in FIG. 4 is substantially at the second end 220 of thetube 200. In such a collecting state, the tube 200 has most of itsinterior surface, preferably in excess of 80% thereof, exposed to thefirst end 210. Moreover, in the collecting state, a route for gas flowfrom the first end 210 via the gauze 250 and through the side tube 240is provided to the bag 100 (not shown in FIG. 3) or directly to ambient;direct venting to ambient is preferred when, for example, screeningtests for less dangerous pathogens are being undertaken.

In the collection state, the user 40 places the first end 210 to his/hermouth so that the ring 260 engages and seals onto the user's lips. Theuser 40 or the tester then activates the assembly 300, for example bydepressing an associated foot pump, to eject the jet 310 of saline mistwhich the user 40 inhales. The inhaled saline mist causes an automaticresponse in the user 40 to exhale forcefully causing air, mucus and/orsputum droplets in the form of a fine mist to be carried from the user's40 lungs into the tube 200. A region of the tube 200 around the secondend 220 forms a low velocity gas region where exhaled air from the useris inclined to flow in a vortex trajectory and thereby deposit its loadof mucus and/or sputum droplets onto internal side-walls of the tube200. Moreover, the user's 40 exhaled air is vented through the side tube240 at a relatively high velocity.

A negative partial pressure may additionally be employed to assist inobtaining the sample.

A sample for analysis is thereby provided on the inside surface of thetube 200, especially in the region of the second end 220. The plunger230 is then actuated relative to the tube 200 for collecting the samplefrom the inside surface of the tube 200 and then for depositing thecollected sample onto an optical interrogation surface of the plunger230 for subsequent optical interrogation and analysis; such actuationalso preferably includes rotation of the plunger 230 relative to thetube 200.

The plunger 230 is thus especially adapted to collecting andmechanically concentrating the sample. Implementations of the plunger230 will now be described with references to FIGS. 4 a and 4 b.

In FIGS. 4 a and 4 b, the plunger 230 is substantially cylindrical inform and comprises a central hollow region 400. The plunger 230 is openat its first end and includes a circular flange 410 for abutting ontothe second end 220 of the tube 200 when the plunger 230 is fullyinserted into the tube 200, thereby limiting an extent to which theplunger 230 can be pushed into the tube 200. The plunger 230 comprisesthe end surface 270 whose plane is substantially perpendicular to acentral longitudinal axis of the plunger 230. At an eccentric region ofthe surface 270 as shown, there is included an optically transmissiveprism 420 extending into the region 400 and a spoon-like projection 430extending outwardly from the surface 270 remotely from the region 400.The spoon-like projection 430 is susceptible in use to scoop-up mucusand/or sputum from the inside surface of the tube 200 thereonto. Thespoon-like projection 430 extends radially at the surface 270 to aperipheral extent of the surface 270. A peripheral edge 440 of theprojection 430 is preferably arranged to slidably contact onto theinterior surface of the tube 200. An optical aperture 450, also known asa prism window, is included in the end surface 270 so that the prism 420is in optical communication with sample sputum and/or mucus collectedonto the projection 430.

The projection 430 is preferably curved over towards its edge remotefrom the peripheral edge 440 as shown in FIG. 3 b to improve performanceof the projection 430 to retain thereon sputum and/or mucus collectedfrom the interior surface of the tube 200.

Moreover, the projection 430 is preferably fabricated from a compliantplastics material and arranged to be bendable to squash its load ofsample material efficiently across the optical aperture 450 when theplunger 230 is advanced fully into the tube 200 and is resilientlypushed against the aforementioned sealing cap (not shown); suitableplastics materials for the projection 430 include one or more ofpolyvinylchloride (PVC), nylon, polytetrafluoroethylene (PTFE),polyethylene, polypropylene, alkylene and silicone rubber. If required,the sealing cap can be provided with a recess for accommodating theprojection 430 so that a flat non-recessed end surface of the sealingcap adjacent to the recess extends to push and spread a buildup ofsample material on a side region of the projection 430 facing towardsthe optical aperture 450 substantially uniformly onto the aperture 450,thereby providing the system 10 with enhanced detection sensitivity. Ifrequired, the thickness of the projection 430 can be reduced to arelative thin neck where it joins onto the end surface 270 to provide aform of hinge so that the projection 430 is tiltable as a flap to squashsample material onto the optical aperture 450.

In order to simplify design of the projection 130 of the reader unit 50,the projection 130 adapted for insertion into the hollow region 400 theplunger 230, it is desirable that the prism 420 is mounted eccentricallybut away from the peripheral extent of the surface 270. When the prism420 and its associated optical aperture 450 are arranged in such amanner, the projection 430 is preferably of a generally “V”-shape formas illustrated in FIG. 4 c. Such a “V”-shape is especially effective atcollecting and retaining a substantial mass of collected sample thereon.Alternatively, as illustrated in FIG. 4 b, the projection 430 can be ofcontinuously curved form; preferably, the projection 430 at its remoteedge is also curved towards the optical aperture 450 nearer towards acentral longitudinal axis of the plunger 230.

The plunger 230 is fabricated as a hollow member so that plunger 230 iscapable of receiving the projection 130 of the reader unit 50 into theregion 400. The projection 130 comprises an electronics module and ispreferably of solid cylindrical form as illustrated in FIG. 5 andindicated generally by 500. Whereas, in use, the sample tube 200 and itsassociated plunger 230 are designed to be disposable items, the readerunit 50 is arranged to be reusable as it comprises moderately costlycomponent parts therein, for example a photomultiplier tube and a diodelaser, which will be described in more detail later. The projection 130is preferably of elongate form comprising a first end 520 and a secondend interfacing onto a module including the display 60. The first end520 comprises an eccentrically-disposed optical interfacing region 510disposed so as to align with the aperture 450, namely the prism window,when the projection 130 is inserted into the hollow region 400 of theplunger 230 to interrogate a sample collected onto the spoon-likeprojection 430.

The sample tube 200, the plunger 230 and the projection 130 are ofadvantage in that they are capable of being compact in storage onaccount of their mutually concentric mountability. Moreover, the tube200 in combination with the plunger 230 is capable of collecting andmechanically concentrating substantially the entire sample exhaled bythe user 40. Furthermore, the plunger 230 is capable of enabling thereader unit 50 to interrogate the sample without coming into contactwith the sample, thereby rendering the reader unit 50 reusable;operational costs are thereby reduced.

The sample tube 200 with its associated side tubes, and the plunger 230are preferably manufactured from plastics materials, for example one ormore of an acrylate, polyethylene, polypropylene, nylon, siliconerubber, polyvinyl chloride (PVC), alkylene, polycarbonate, andpolytetrafluoroethylene (PTFE) plastics materials. More preferably, atleast one of the tube 200 and the plunger 230 are injection molded.Alternatively, one or more of the sample tube 200 and its associatedside tubes, and the plunger 230 can be fabricated from extruded metalsheet, or even fabricated by die-cast metal techniques.

Most preferably, the sample tube 200, the plunger 230 and its associatedport 240 and filter 250 are molded as a single component part. Likewise,the plunger 230 with its associated projection 430 and prism 420 arepreferably molded as a single component from a substantially opticallytransparent plastics material, for example a polycarbonate or acrylicplastics material. Alternatively, the plunger. 230 can be fabricatedfrom a substantially black plastics material, for example PVC, and theprism 420 subsequently assembled thereinto; the use PVC is of advantagein shielding the prism 420 from stray ambient illumination, and alsoshielding the remote end of the projection from ambient illuminationwhen inserted into the plunger 230 during measurement.

At the end surface, the plunger 230 can optionally include a smallorifice, for example a substantially round orifice having a diameter ina range of 0.1 mm to 2.5 mm. This orifice is of advantage for injectingan atomized spray mist, for example a saline mist, into the sample tube200 when deployed to collect a sample of mucus and/or sputum from theuser 40. Injection of such a mist into the tube 200 prior to the user 40exhaling the sample for collection onto inner surfaces of the tube 200is of benefit in obtaining substantial quantities of mucus droplets fromthe user 40. For other types of bioassay, it is found that the additionof a small quantity of liquid, for example a saline or a buffersolution, is desirable in that it assists the diffusion of microbes, forexample bacteria, to be tested towards the optical aperture 450. Suchliquids can be added to the system 10 either before or after samplecollection, as appropriate using, for example, an aerosol spray ordroplets from a pipette.

The tube 200, the plunger 230 and the projection 130 are advantageouslyfabricated to be within preferred size ranges. For example, the sampletube 200 preferably has a diameter in a range of 20 mm to 30 mm.Moreover, the side tube 240 preferably has a diameter in a range of 1 mmto 10 mm, more preferably in a range of 5 mm to 8 mm. Furthermore, thesample tube 200 preferably has a length in a range of 40 mm to 150 mm,more preferably in a range of 50 mm to 80 mm. The optical aperture 450preferably has an area in a range of 9 mm² to 64 mm². It will beappreciated that these dimensions are appropriate for the system 10designed for use with human subjects. Other species will require thesedimensions to be appropriately modified.

The sample tube 200 and its associated plunger 230 are preferablyprovided with a locking mechanism such that when the mucus and/or sputumsample has been mechanically concentrated within the tube 200 anddelivered efficiently onto the optical aperture 450 and the plunger 230moved to its measurement position, the plunger 230 is mechanicallylocked into position relative to the sample tube 200. Such a mechanismis of advantage in that it is capable of preventing the sample tube 200and its plunger 230 being reused; in poorer parts of the world, there isa temptation to reuse medical parts, for example syringes. Morepreferably, insertion of the projection 130 of the reader unit 50triggers engagement of such a mechanism to prevent reuse. A temptationfor reuse can potentially occur where the system 10 displays anon-positive indication for the presence of a pathogen. Such a lockingmechanism is of further advantage in that locking of the sample tube 200to the plunger 230 together with the sealing cap forms an enclosedregion for isolating dangerous pathogens. Yet more preferably, thesample cap also snap engages onto the tube 200 so that the two cannotsubsequently be disengaged.

In order to enhance a vortex generated within the sample tube 70, 200and thereby improve sputum and/or mucus deposition therein, the tube 70,200 can be provided with additional features to modify air flow therein.Referring to FIG. 6, there is shown a modified sample collection tubeindicated generally by 600, the tube 600 including an annular septumorifice 610 included between the atomizer assembly 300 and the side tube240. The septum 610 is preferably included as near as possible to theend 210. Moreover, the septum orifice 610 is preferable molded to be anintegral part of a cylindrical part 620 of the tube 600. The projection430 of the plunger 230 is preferably made flexible so that advancing theplunger 230 into the tube 600 after sample collection therein causes theprojection 430 to flex against the septum orifice 610 to squash thesample onto the optical aperture 450.

The septum orifice 610 assists by enhancing peripheral drag to generateeddies and corresponding complex multiple vortex formation, therebyenhancing mucus and/or sputum deposition on an inner surface of thecylindrical part of the tube 600. Moreover, the orifice 60 also assiststo prevent saline mist encroaching into regions of the tube 600 remoterfrom the user 40 in use. Preferably, the septum orifice 610 is recessedbehind the end 210 by a distance in the order of 5 mm to 15 mm.Moreover, the septum orifice 610 preferably includes a central holehaving a diameter in a range of substantially 3 mm to 20 mm. The septumorifice 610 is preferably fabricated from a collapsible material (forexample a flexible plastic) in order that the sample collected on thewalls of the sample tube 200, closest to the user's 40 mouth, can beconcentrated onto the optical aperture 450.

The sample tube 200 can alternative be modified to include a directionalbend near the opening 210 to generate a sample collection tube indicatedgenerally by 650. The assembly 300 is preferably included on an outsidebend portion of the tube 650 as illustrated to inject saline misttowards the end 210. The directional bend causes an asymmetricalspatially varying drag to air that promotes eddy formation.

In order to obtain superlative collection performance, a combination ofthe features of the tubes 600, 650 can be employed.

Further alternative embodiments of sample collection unit 30 arepossible.

For example, in FIG. 7, there is shown a sample collection chamberindicated generally by 700. The chamber 700 comprises an inlet pipe 720of substantially 25 mm diameter for delivering exhaled breath from theuser 40 to a collection box 710. More preferably, the inlet pipe 720 hasa diameter in a range of 18 mm to 30 mm. The collection box 710 includesat its periphery a prism 750 susceptible to promoting evanescentradiation propagation at an exposed surface thereof facing inwardly intothe box 710 where mucus and/or sputum deposition occurs from the exhaledbreath. Sample collection onto the exposed surface is promoted by avortex generated within the box 710; this vortex is especially enhancedwhen an exit pipe 760 from the box 710 has a diameter which is less thanthat of the inlet pipe 720. Preferably, the diameter of the exit pipe760 is substantially 5 mm, although a diameter in a range of 2 mm to 10mm is especially preferred. The prism 750 can be situated in anyposition on the walls of the box 710 in order to collect samplesthereon.

Exhaled breath from the box 710 is beneficially conveyed along the exitpipe 760 to the bag 100 and its associated gas exit orifice 105.Alternatively, the exhaled breath output from the box 710 can beinitially passed through a filter to remove pathogens and then vented toambient or to the bag 100. Beneficially, a venturi can be incorporatedinto the inlet and/or exit pipes to assist with vortex formation withinthe box 710. The exit pipe 760 and inlet pipe 720 can be placed at anyposition relative to each other and the prism 750, within the box 710.

The projection 130 of the reader unit 50 will now be further describedin more detail with reference to FIGS. 8 a and 8 b. In order to obtainoptimum readout from the prism 420, optical interrogation components ofthe reader unit 50 are preferably housed within the assembly. Theprojection 130 therefore comprises a photomultiplier tube (PM tube) 800and a diode laser. Advantageously, the PM tube 800 is a proprietarydevice manufactured by Hamamatsu Photonics K.K. of Japan, the devicehaving a part number R7400U-01. A photosensitive face of the PM tube 800is orientated towards the optical interface region 510, preferablyspatially as close thereto as possible. Moreover, the projection 130further comprises a solid state diode laser 810. A configurationdepicted in FIG. 8 a is most preferred as it results in least opticallosses when coupling optical radiation to the optical aperture 450.However, especially when the projection 130 is of a relatively smallexterior diameter, it is convenient for the diode laser 810 to becoupled via a light guide 820, for example comprising a parallel bundleof optical fibre waveguides. In a standard relatively larger version ofthe projection 130 illustrated in FIG. 8 b, the diode laser 810 and thePM tube 800 are mounted mutually adjacently, thereby requiring only arelatively shorter length of light guide to be employed. However, in acompact relatively smaller version of the projection 130 depicted inFIG. 8 b, the diode laser 810 is positioned behind the PM tube 800 asillustrated and a relatively longer section of light pipe 830 employedto convey light from the diode laser 810 to the interface region 510. Ifrequired, the projection 130 can be fabricated from diecast, machined orextruded metal and the diode laser 810 can be thermally coupled to theperipheral wall of the projection 130 for cooling purposes. Similarthermal considerations pertain to the PM tube 800 although this devicedissipates relatively negligible power in operation. Design of thereader unit 50 comprising the projection 130 and its associated partswill be described in more detail later.

Referring again to FIG. 2, the aforementioned sealing cap applied to thecollection tube 70 is denoted by 900. The cap 900 is illustrated incross-section in more detail in FIG. 9, the cap 900 comprising a mainbody component 905, first and second liquid reservoirs 910, 920including liquid masses 930, 940 respectively, and a sealing top 950hermetically bonded to the body component 905 to seal the liquid masses930, 940 into the cap 900. The body component 905 and the sealing top950 are preferably fabricated from a highly flexible plastics material,for example soft silicone rubber. The sealing top 950 is substantially arelative thin flexible membrane including domed regions 960, 970 alignedto corresponding reservoirs 920, 910 respectively. Bonded centrally tothe domed regions 960, 970 are steel pins 980, 990 respectively. Thesteel pins 980, 990 have blunted broadened ends where they are moldedinto the sealing top 950, and sharp pointed ends where they face towardsend regions of the reservoirs 920, 910. The sealing cap 900 furthercomprises a retaining feature 1000, for example a barbed insert withbackwardly directed barbs which bind into the collection tube 70, 200 toallow the cap 900 to be easily inserted into the tube 70, 200 but notremoved therefrom again.

In operation, the user 40, or preferably the tester, depresses the domedregions 960, 970 to cause the pins 980, 990 to puncture their respectivereservoirs 920, 910 to release the contents of their respective liquidmasses 940, 930 into the sample tube 70, 200 to chemically process mucusand/or sputum collected at the optical aperture 450 of the plunger 110,230.

In manufacture, the body component 905 is orientated so that its endface 1010 is downwardly facing. The reservoirs 910, 920 are then filledwith their respective liquid masses 930, 940 and then the sealing top950 comprising its pins 980, 990 is ultrasonically welded, or otherwisehermetically bonded, into a recess molded into the body component 905 asillustrated.

Although two reservoirs 910, 920 are shown in FIG. 9, it will beappreciated that the sealing cap 900 can be fabricated to have one ormore reservoirs. Moreover, the liquid masses 930, 940 can be varied incomposition depending upon the type of pathogen to be detected by thesystem 10. For example, one of the liquid masses can be a biologicalbacterial lysing agent whereas another of the liquid masses can be abiological fluorescent marker agent. These agents will be described inmore detail later.

3.2 Reader Unit

The reader unit 50 illustrated in FIG. 1 will now be described in moredetail.

Referring to FIG. 10, there is shown an optical configuration indicatedgenerally by 1100. This configuration 1100 is employed in the system 10and its parts are distributed between the reader unit 50 and itsassociated projection 130, and the plunger 110, 130.

In particular, the reader unit 50 in its projection 130 includes thelaser 810, the light guide 820, 830 (if required), an optical filter1120, the PM tube 800 (and associated power supply, not shown) and aminiature solid-state diode detector 1130. A main part of the readerunit 50 includes the display 60, a microcontroller 1150 for executingcalculations, a synchronous demodulator 1140, and a strobe circuit 1160.

The plunger 110, 230 comprises a prism 420; in particular, the preferredprism 420 is a dove-type prism of a cross-sectional trapezoidal form asillustrated. A major front face of the prism provides the opticalaperture 450 that is coated in a biologically active layer 1110 whichwill be described in further detail later.

Component part interconnection within the configuration will now bedescribed with reference to FIG. 10. The microcontroller 1150 includes adata output which is coupled to the display 60. In its simplestconfiguration, the display 60 merely includes a yes/no indicator forindicating whether or not a given pathogen is present in the mucusand/or sputum sample above a predefined threshold. In a more complexconfiguration, the display 60 provides a quantitative measure of theconcentration of pathogens present in the mucus and/or sputum samplesbeing interrogated; the display 60 can be one or more of a liquidcrystal display (LCD), for example an alpha-numerical LCD display, alight emitting display (LED) and a miniature plasma display. Themicrocontroller 1150 is also connected at its output to the strobecircuit 1160 for modulating power applied to the laser 810, therebytemporally modulating its optical output beam 1200 launched into thelight guide 820, 830. Moreover, the microcontroller 1150 also includesan input S₃ for receiving a demodulated output signal from thesynchronous demodulator 1140. The PM tube 800 includes a signal outputS₁ which is connected to a signal input of the synchronous demodulator1140. Furthermore, the diode detector 1130 includes a signal output S₂which is coupled to a strobe input of the demodulator 1140. The opticalfilter 1120 is included between the PM tube 800 and a smaller majorrear-plane face 1170 of the prism 420 as illustrated; the filter 1120 iseffective at transmitting radiation components arising from evanescentwave interaction in the active layer 1110 and reflecting and/orabsorbing scattered radiation generated directly from scatter of primaryradiation within the prism 420.

Operation of the optical configuration will now be described withreference to FIGS. 1 and 10. The user 40 activates the system 10 whichcauses the microcontroller 1150 in combination with the strobe circuit1160 to generate a modulated signal to drive the diode laser 810 andthereby generate the correspondingly strobed beam 1200. Preferably, thewavelength of radiation output from the laser 810 will be selected tomatch the excitation wavelength of fluorophores employed to analyse themucus and/or sputum sample collected onto the optical aperture 450. Suchfluorophores can be selected to be selectively responsive at differentwavelengths, for example deep red, green, or blue radiation wavelengths.The use of longer wavelength radiation from the laser 810 correspondingto red radiation is preferable to reduce costs as red lasers are highlyinexpensive and readily available. Conversely, solid state laser diodescapable of outputting radiation at relatively shorter blue and greenradiation wavelengths are presently relatively expensive. However, a PMtube that is sensitive in the red spectral region must be used and theseare slightly more expensive than those most sensitive in the blue/greenregion. However, the optical configuration 1100 is, therefore, capableof being operated at different wavelengths to match the type offluorophores employed to analyse the mucus and/or sputum sample, and thesystem 10 can be constructed to work at any optical frequency desired.

The beam 1200 is preferably strobed at a frequency in a range of 100 Hzto 100 kHz. More preferably, the beam 1200 is strobed at a frequency ina range of 100 Hz to 1500 Hz. Most preferably, the beam 1200 is strobedat a frequency of substantially 1030 Hz as this renders amplifiercircuits (not shown) associated with the PM tube 800 and the synchronousdemodulator 1140 straightforward to design using standard components asbandwidth constraints are not especially problematic at such a strobefrequency. Moreover, 1030 Hz is not a harmonic of 50 Hz mains supply,thereby rendering the system 10 less susceptible to be affected by 50 Hzfluctuating light sources such as mains-operated fluorescent striplights frequently found in hospitals and clinics.

Preferably, the strobe circuit 1160 is operable to modulate theinjection current used to excite the laser 810 so that this current isperiodically switched above and below the lasing current threshold ofthe laser 810. Alternatively, the laser 810 can be operated at constantoutput intensity and a separate modulator device, for example a liquidcrystal (LCD) cell, used to temporally modulate an output beam from thelaser 810 to generate the radiation beam 1200.

The laser 810 emits the strobed beam 1200 which propagates through thelight guide 820, 830 to generate a corresponding exit beam 1210 whichpropagates to a first inclined face 1215 of the prism 420 and isrefracted thereat to generate a corresponding refracted beam 1220 whichsubtends an angle θ relative to a normal to the plane of the opticalaperture 450. Preferably, the angle θ is in a range of 62° to 80°. Morepreferably, the angle θ is substantially 70°.

The refracted beam 1220 propagates to the optical aperture 450 and ismostly reflected thereat to generate a reflected beam 1230 that thenpropagates to a second inclined face 1235 of the prism 420 to emergerefracted therefrom as a beam 1240 which then propagates to the detector1130. The beam 1240 gives rise to a strobe signal at the output S₂ whichis used as a modulation reference signal for the demodulator 1140.

Where the beam 1220 impinges onto the optical aperture 450, a fractionof the radiation present in the beam 1220 is coupled into the plane ofthe aperture 450 in the form of an evanescent wave 1245. This evanescentwave 1245 propagates in a boundary region at the interface of thebiologically active layer 1110 to the prism 420 itself. The boundaryregion is frequency dependant and at the frequencies in which the systemworks this is effectively only in the order of 100-200 nm thick. Thus,coupling of the beam 1220 to form the evanescent wave 1245 allows forextremely efficient optical interrogation of chemicals present at theboundary region. If fluorophores are present at the boundary region,they are excited by the evanescent wave to generate fluorescentradiation. Preferably, this fluorescent radiation is at a differentradiation frequency to that of the beam so that the filter 1120 can beused to discriminate scattered radiation from the beam 1220 fromfluorescence at the aforementioned boundary region; namely, fluorophorespresent at the boundary region are operable to provide radiationwavelength conversion.

Fluorescent radiation generated at the boundary region propagates fromthe boundary region through the prism 420 to exit from the prism face1170 and propagate through the filter 1120 to the PM tube 800 to cause acorresponding sense signal to be generated at the output S₁. The sensesignal passes to the signal input of the demodulator 1140 and issynchronously demodulated therein with respect to the signal from theoutput S₂ to provide a demodulated signal at the output S₃ which passesto the microcontroller 1150 for subsequent sampling and conversion tocorresponding data D. The microcontroller 1150 then proceeds to comparethe data D with a preprogrammed threshold level T and determine therebywhether or not pathogens are present in the mucus and/or sputum samplescollected onto the optical aperture 450 and interrogated by theevanescent wave radiation propagating therealong.

Preferably, the degree fluorescence, namely the magnitude of the data D,can be determined prior to, namely providing data D1, and then againafter, namely providing data D2, collecting and mechanicallyconcentrating the sample of sputum and/or mucus onto the opticalaperture 450. A difference value given by Equation 1 (Eq. 1):ΔD=modulus(D ₂ −D ₁)  Eq. 1is then calculated in the microcontroller 1140. This difference value ΔDis then compared with the threshold value T to determine whether or notpathogens are present in the sample. Such a difference method ofmeasurement is effective at removing systematic contributions to thesense signal provided at the output S₁; such systematic contributionscan arise from scatter within the prism 420, residual fluorescencewithin the prism 420 especially if it is fabricated from plasticsmaterials, and finite radiation wavelength discrimination provided bythe filter 1120. Suitable plastics materials for fabricating the prism420 include perspex, acrylate, polycarbonate and polymethylmethacrylate(PMMA). It should be noted that the use of polymer materials thatexhibit fluorescence is be avoided where possible.

Preferably the threshold value T is made proportional to the opticalinterrogation radiation power delivered into the beam 1210 from thelaser 810. More preferably, the threshold value T is made proportionalto the radiation power in the beam 1240 received at the detector 1130 soas to account for efficiency of optical coupling into the prism 420which can potentially vary from plunger 230 to plunger 230, especiallyif mechanical tolerances in manufacture are not tightly controlled.

Although the use of the PM tube 800 is described in the foregoing, itwill be appreciated that other types of optical detectors canpotentially be employed, for example avalanche photodiodes,phototransistors or low-noise photodiodes. If signal-to-noiseconsiderations allow, the laser 810 is preferably substituted with alower-cost high-brightness light emitting diode (LED).

If required, the filter 1120 can comprise several optical filtercomponents to enhance its wavelength discrimination, for example byutilizing several diffraction grating layers. Moreover, themicrocomputer 1150 can be programmed to account for systematic steadytemporal drift in the sense signal to account for warm-upcharacteristics of the system 10 when activated from a cold state. Anestimate of such drift can be made by interrogating the optical aperture450 for a period of a few minutes before introducing the mechanicallyconcentrated sample thereto.

Preferably, the microcontroller 1150 includes a data logger forrecording test results and corresponding reference codes for subsequentdownloading to a database from the reader unit 50. In such aconfiguration, the reader unit 50 preferably includes a data entry keypad so that each test performed by the reader unit 50 can be allocatedan identification reference. When the microcontroller 1150 is configuredto provide a data logging characteristic, the microcontroller 1150 canpotentially be used effectively during a disease epidemic to generatepathogen infection rate statistics.

It will be appreciated that the system 10 can be adapted forinterrogating liquid samples from other sources than exhaled breath.Referring to FIG. 11, there is shown a alternative configuration forpart of the system 10 for analysing a liquid, for example a bloodsample, flowing or stagnant within a tube 1310. The prism 420 is anintegral part of, or is attached to, a side region of the tube 1310. Thebeam 1210 passes through the prism 420 as the beams 1220, 1230 andexcites fluorophores attached to the major face of the prism 420 facingin contact towards the liquid sample within the tube 1310. Fluorescenceof the fluorophores in response to composition of liquid sample, forexample blood, is received by the PM tube 800 to generate a strobe sensesignal at the output S₁ for subsequent synchronous detection. The system10 modified according to FIG. 11 is thus susceptible to providecontinuous monitoring of blood or other body fluids, for example urine,for pathogens and therefore has widespread potential application inhospitals and body fluid processing facilities.

Moreover, if required, a sample air stream can be continuously passedthrough the tube 1310 to detect for air-borne pathogens, toxicpollutants and explosive vapours for example. Thus, the biologicalmeasurement system 10 is potentially adaptable to other applicationsother than merely to detect respiratory pathogens such as tuberculosis.

The dove-type prism 420 can be substituted in the plunger 110, 230 withan alternative prism indicated generally by 1400 in FIG. 12. The prism1400 preferably has internal angles of 55°, 125°, 70° and 110° asillustrated. Optionally, a face indicated by 1410 can be a mirror coatedsurface to enhance reflective performance of the face 1410 whenreflecting the beam 1210 to form the beam 1220. By reducing internalreflection losses, the prism 1400 is potentially capable of imparting anenhanced detection signal-to-noise ratio to the system 10.

Furthermore, dove-type or other prisms where the angle of acceptance issuch that multiple reflections are induced within the prism can beadopted. For example, suitable alternative prisms are described in abook by C. N. Banwell and E. M. McCash, “Fundamentals of MolecularSpectroscopy” (1994) McGraw-Hill, 4^(th) edition which is herewithincorporated by reference.

It should be appreciated that fluorimetry has been shown to be ofconsiderable importance for the detection of biological materials suchas proteins and DNA, where fluorophores on antibodies are used asmarkers for detection. Detection using such fluorimetry can be executedby way of either

-   (a) bulk fluorescence, measurements; or-   (b) through the application of interrogation techniques such as    evanescent wave detection; or-   (c) cavity ring down spectroscopy; or-   (d) through the use of displacement assays.

Such fluorimetry offers some potential advantages in terms ofspecificity, simplicity, and sensitive. Evanescent wave detection iswell known, but low-cost evanescent wave fluorimeters are not yetcommercially available for use in pathogen detection as described withrespect to the present invention.

It should be further appreciated that sample analysis followingcollection onto an optical interrogation area can be undertaken usingmeans other than fluorimetry, for example, radioactive andphosphorescent markers can be utilized or chemiluminescence techniques.

Detection can also be carried out using other forms of spectroscopy, notassociated with immunoassay systems or evanescent waves. For example,infrared spectroscopic methods can identify the presence of specificmolecular fragments on the basis of ‘group frequencies’ at specificregions of the infrared spectrum; these can be performed using bothtransmission and reflection geometries where the latter detects theabsorption of evanescent IR radiation. Other possibilities arepreferably, but not exclusively, Surface Acoustic Wave (SAW) detectionand Surface Plasmon Resonance (SPR) which may provide signal enhancementand thus gains in sensitivity.

4. System Biochemistry

In the foregoing, the measurement system 10 is described with respect toits hardware. In the following description, chemical aspects of thesystem 10 will now be elucidated in more detail.

4.1 Biochemical Overview

The measurement system 10 is capable of operating according to twoalternative detection methods, namely either:

-   (a) by fluorophore displacement resulting from the presence of a    pathogen (namely competitive displacement assay); or-   (b) by fluorophore binding promoted by the presence of a pathogen    (namely selective binding assay).

In the competitive displacement assay, the sense signal at the output S₁reduces as the pathogen is introduced into the sample collection unit30. Conversely, in the selective binding assay, the sense signal at theoutput S₁ increases as the pathogen is introduced into the collectionunit 30. Both assays are pertinent as certain types of pathogen are bestdetected by one or other of the assays.

In the selective binding assay, the optical aperture 450 is coatedduring manufacture with a first antibody that will bind to the pathogento be detected. In operation, the pathogen is mechanically concentratedonto the optical aperture 450 and becomes immobilized thereat on accountof its affinity to the first antibody. Next, fluorophores bound tosecond antibodies having affinity to the pathogen are released into thesample tube 70, 200 so that the fluorophores become bound to thepathogen immobilized to the first antibodies at the optical aperture450. If required, the first and second antibodies can be identicalalthough this is not essential as pathogens frequently exhibit severalsurface regions to which different antibodies can bind. The secondantibodies and their associated fluorophores can be in the form of aliquid held in one of the reservoirs 910, 920 of the sealing cap 900.When the pathogen has been immobilized directly to the first antibodiesat the optical aperture 450 together with the fluorophores bound to thesecond antibodies immobilized to the pathogen, the evanescent waveradiation 1245 is capable of interacting strongly with the fluorophores,thereby generating significant fluorescence for detection by the PM tube800.

In the competitive binding assay, the optical aperture 450 is coatedduring manufacture with the first antibody that will bind the pathogenbeing investigated. Moreover, during manufacture, fluorophores bound toanalogues of the pathogen that bind weakly to the first antibody areadded to the optical aperture 450. When the mechanically concentratedsample is applied to the aperture 450, the pathogen therein displacesthe weakly bound fluorophores and associated analogues and bind insubstitution to the immobilized first antibodies. The weakly boundfluorophores and associated analogues, when displaced, migrate away fromthe boundary region supporting the evanescent wave propagation 1245causing a decrease in fluorescence detected by the PM tube 800.Preferably, one or more of the reservoirs 910, 920 of the sealing cap900 includes a wash agent to assist removal of the displacedfluorophores bound to associated analogues from the optical aperture 450so that a final settled reading is more rapidly attained.

It should be noted that the antibodies that can be used for these testsmay be monoclonal or polyclonal in form.

4.2 Antibody Immobilisation

The immobilisation of antibodies to glass or plastic surface, forexample to the optical aperture 450, is already well studied, and manyprotocols exist. These protocols derive largely from the success ofknown ELISA tests, in which antibodies immobilised on a 96-well plateform a crucial component of such tests. In a textbook “mobilizedBiomolecules in Analysis: a practical approach”, edited by T. Cass andF. S. Ligler (Oxford University Press), said textbook herewithincorporated by reference, there is provided a thorough overview of manyimmobilisation protocols.

Such protocols will each typically comprise:

-   (a) a preparation step, in which a surface is cleaned and optionally    activated;-   (b) an incubation step during which antibodies or antibody fragments    are attached to the surface; and then-   (c) a blocking step to prevent further non-specific binding of    biomolecules to the surface

Rinsing steps generally follow the incubation and blocking steps. Theprepared surface is then dried and stored in a dry atmosphere.Incubation times depend to a significant extent on the molecule (namelypathogen) to be captured and the surface composition.

Activation of the surface is commonly achieved by irradiating thesurface, or exposing it to plasmas or chemicals such as silanesincorporating an active group to which antibodies can be bound. Oncechemically active groups exist on the surface, a simple incubation stepis usually sufficient to bind the antibodies, such antibodies alsoreferred to in the following as receptors.

4.3 Analogues for the Competitive Binding Assay

Analogues used in the aforementioned competitive displacement assaycorrespond to molecules or molecular groups which bind to receptors, forexample antibodies, immobilised on the optical aperture 450 of the prism420, but do so with a lower association constant than the pathogen to bedetected. Preferably, the analogue-receptor association constant is lessthan 10% of the pathogen-receptor association constant, and if possibleless than 1%.

These analogues may be similar molecules from closely related species,for example sheep luteinising hormone is capable of providing ananalogue of human chorionic gonadotropin. Alternatively, the analoguescan be molecules synthesised to mimic the structure of the pathogen,especially the epitope to which the antibody binds, or a modifiedversion of the pathogen.

It is also potentially advantageous to use derivatives of the pathogenas analogues. Such derivatives can be artificial derivatives such asmolecules modified by adding bulky or ionic groups (which can reduce thebinding energy), by adding steric or charge interference, or binding toa bulky group, which can cause a conformational change in the bindingsite of the pathogen. In the case of analytes which are proteins, thecorresponding amino acid sequence of the protein can be modified nearthe binding site to the receptor by recombinant molecular biologytechniques, such as site directed mutagenesis. Alternatively they can benatural metabolites of the target analyte.

Techniques required to prepare such analogues are known, and there ismuch prior art on the design of such analogues. Chemical modificationsof organic molecules, biochemical modifications of naturally occurringmolecules and synthesising structural mimics or molecules are known inthe art. The suitability of a candidate analogue can be determined by acompetitive ELISA assay between the analyte and the candidate analogueor by a measurement of the association constant with the receptor.

Polyclonal and monoclonal antibodies reactive against pathogens such asmycobacterium tuberculosis are readily available from several commercialvendors, for example Skybio Ltd. in the United Kingdom. Such antibodiescan be purchased in sizeable batches and labeled and immobilized usingstandard known chemistries.

4.4 Fluorophores

Selection of suitable fluorophores for use in the system 10 has animportant bearing on the technical performance of the system 10, forexample its signal-to-noise ratio and hence its ability to identifyearly onset of disease.

There are many commercially available fluorophores. The most significantqualities of such fluorophores are:

-   (a) their absorption band, which limits the range of interrogating    radiation wavelengths at which they can be excited; and-   (b) their emission band, namely the range of wavelengths over which    fluorescent radiation is emitted from the fluorophores when excited.

The absorption band must overlap as much as possible with the spectrumof the interrogating light source used, namely the laser 810 in thesystem 10; moreover, the emission band should overlap as little aspossible with the absorption band. These qualities limit the range offluorophores which can be used in the system 10. Other factors which mayinfluence the choice of an optimal fluorophore for the system 10 are:

-   (a) the ease with which the fluorophore can be coupled to a    corresponding target molecule, for example an antibody or analogue;    and-   (b) the separation between the absorption and emission bands of the    fluorophore; and-   (c) the brightness of the fluorescent radiation emitted from the    fluorophore.

A commercial company Molecular Probes manufactures and supplies acommercial range of fluorescent dyes called the Alexa dye series. Thisseries includes a number of bright fluorophores with optimal excitationwavelengths ranging from 346 nm to 684 nm, including many moleculesspecifically designed to work well with common light sources such asbright laser diodes or red LEDs. Many other dyes exist and are widelyused, for example fluorescein isothiocyanate (FITC), BODIPY,phycoerythrin, allophycocyanin (APC), rhodamine, Texas Red and OregonGreen. Some of these dyes and their relevant parameters are listed inTable 1; one or more these dyes can, if required, be employed in thesystem 10 either alone or in combination.

TABLE 1 Examples of typical fluorophores susceptible for use in thesystem 10. Absorption Emission peak peak Dye name Abbreviation (nm) (nm)Fluorescein isothiocyanate FITC 493 520 R-phycoerythrin RPE 495, 536 576B-phycoerythrin BPE 546 576 Rhodamine — 550 573 Rhodamine B — 578 604Allophycocyanin APC 630, 645 655, 660 Alexa Fluor 350 — 346 442 AlexaFluor 430 — 433 539 Alexa Fluor 488 — 495 519 Alexa Fluor 532 — 532 554Alexa Fluor 594 — 590 617 Alexa Fluor 633 — 632 647 Alexa Fluor 680 —684 707 BODIPY 493/503 — 500 506 BODIPY 665/676 — 665 676 Cy5 — 649 666,670 Texas Red — 595 620 Teramethyl rhodamine TRITC 550 573isothiocyanate

In order to enhance performance, latex spheres including fluorescentmaterials can be bound to one or more of antibodies and analogues inorder to provide the system 10 with enhanced detection sensitivity. Suchlatex spheres are capable of exhibiting an enhanced degree offluorescent radiation in response to being excited by the evanescentradiation wave 1245 at the optical aperture 450.

Latex spheres are commercially available from companies such as DynalBiotech with a wide range of surface chemistries; such surfacechemistries can include fluorophores and also impart the spheres withmagnetic properties. In the sample tube 70, 200, magnetic attraction oflatex spheres comprising fluorophores when mechanically concentratingthe sputum and/or mucus sample onto the projection 430 is highlyadvantageous to achieving enhanced measurement sensitivity from thesystem 10. The latex spheres used for this system are preferably in therange of 50 nm to 1 μm in diameter; more preferably, the spheres are inthe range of 100 nm to 200 nm in diameter, namely in line with theboundary depth of the evanescent wave penetration at the opticalaperture 450.

In the selective binding assay, magnetically labeled fluorescent latexspheres can be released from one of the reservoirs 910, 920 of thesealing cap 900 into the sample tube 70, 200 prior to mechanicalconcentration of the sample at the optical aperture 450; preferably, theaperture 450 and/or the projection 430 are provided with one or more,small, movable, permanent magnet(s) thereat to assist latex spherecollection. Following collection and concentration of the sample ontothe optical aperture 450, the magnet(s) can be moved away to allow thespheres that have not been chemically bound to the aperture 450 todiffuse away into the bulk liquid, leaving only the bound species to bedetected at the aperture 450.

4.5 Optional Lysis Sensitivity Enhancement

Sensitivity of the system 10 to the detection of pathogens can beenhanced by employing a process known as lysis. Lysis is the process ofbreaking cells, for example pathogen microbes, into its componentfragments. Antibody-labeled fluorophores are capable of binding to thesefragments. Moreover, the fragments are susceptible to binding toantibodies at the optical aperture 450.

There are a wide variety of methods used to lyse microbes, for examplebacteria. Such methods comprise one or more of chemical, mechanical andthermal processes. These processes are known to the skilled addressee.Lysis of pathogens collected within the sample tube 70, 200 is ofadvantage in that lysis fragments are susceptible to binding to firstantibodies immobilized at the optical aperture 450 and also to secondantibodies bound to associated fluorophores. Thus, lysis is capable ofenhancing the detection efficiency in the aforesaid selective bindingassay within the system 10, for example by at least an order ofmagnitude. Likewise, lysis is capable of giving rise to more competitivedisplacement sites at the optical aperture 450 in the case of theaforesaid competitive displacement assay.

Chemical methods of lysis involve the use of enzymes such as lysozyme,or detergents such as SDS to break down cell walls. Mechanical methodsphysically break down cell membranes; examples of mechanical methodsinclude nitrogen cavitation bombs, french press or hughes press,sonication, glass beads or osmotic lysis techniques. Thermal lysisemploys extremes of temperature excursions to destroy cell walls; suchtemperature excursions can comprise repeated freezing and thawing of acell culture.

Mycobacteria, for example mycobacterium tuberculosis, are especiallydifficult to lyse. Lysis buffers specifically adapted for mycobacteriacomprise additional reagents such as lysozyme to break down mycobacteriacell walls.

During lysis, enzymes released from cell interior regions often attackmolecules of interest for detection purposes within the system 10.However, lysis buffers can be formulated to include additionalingredients such as protease inhibitors to prevent the target moleculefrom being digested or denatured. Table 2 provides a list of lysisprotocols susceptible for use within the system 10 to enhance itspathogen detection performance.

TABLE 2 Examples of lysis protocols Principles Reference Method usedClass of target Gen-Probe package insert Sonicate for 15 minutes inMechanical, Mycobacterium lysis buffer and glass beads chemical Pierreet al., J. Clin. Micro. 15 minutes at 95° C. with Thermal, Mycobacterium29 (4): 712-717 (1991) 0.1M NaOH, 2M NaCl, chemical 0.5% SDS Hurley etal, Int. J. 3 minutes in minibead Mechanical Mycobacterium SystematicBacteriology 38 beater with distilled phenol (2): 143-146 (1988) andzirconium beads Robson et al., U.S. Pat. No. Heating for 2 to 15 minutesThermal Mycobacterium 5376527 at 60° C. to 100° C. Pierce productinformation Shake sample with B-PER Chemical Bacterium Bacterial ProteinExtraction Reagent for 10 minutes

Lysis is preferably performed in the collection apparatus 30 eitherbefore mechanical concentration of the sample has occurred therein orafter mechanical concentration has been achieved.

It will be appreciated that sensitivity of the system 10 can be furtherenhanced by utilizing many known amplification techniques employed instandard immunoassay. Such amplification techniques include, but are notlimited to, biotin/axidine or biotin/streptavidin sandwich techniquesand enzyme-linked assays. Also, chromogenic substances can be used insubstitution, or in addition to, the fluorophores, for example as inELISA assays, producing a colour change in solution rather thanfluorescent signal as in the system 10 described above. Such colourchange can be detected electronically using colour sensitive electronicdetectors or using the naked eye.

4.6 Description of Biochemical Interactions within the System

In order to more completely describe operation of the system 10,especially with regard to biochemical reactions occurring therein,reference will be made to FIGS. 13 to 15.

4.6.1 Selective Binding Assay

Referring to FIG. 13, there is illustrated a binding process whichoccurs in operation within the sample tube 200 at the optical aperture450.

In STEP A, the first antibodies denoted by 1500 are bound to the opticalaperture surface 450, the first antibodies 1500 deposited duringfabrication of the plunger 110, 230. The optical surface is opticallyinterrogated with evanescent wave radiation and a first degree offluorescence measured.

In STEP B, the mucus and/or sputum sample is mechanically concentratedwithin the sample tube 70, 200 as described in the foregoing anddeposited at the optical aperture 450 whereat specific pathogens 1520 ofinterest bind to the first antibodies 1500 as illustrated.

In STEP C, the fluorophore-labeled second antibodies 1530, 1540 arereleased from one or more of the reservoirs 910, 920 of the sealing cap900 by rupturing them as described earlier with reference to FIG. 9; thefluorophore-labeled antibodies 1530, 1540 wash onto the optical surface450 as in STEP B and bind to the specific pathogens 1520 in STEP C. Theoptical aperture 450 with its bound first and second antibodies, 1500,1530, pathogen 1520 and fluorophores 1540 can then be opticallyinterrogated using evanescent wave radiation of identical magnitude asused to determine the first degree of fluorescence, thereby enabling asecond degree of fluorescence to be measured. A difference between thefirst and second degree of fluorescence gives an indication of thepresence of the fluorophores 1540 from which can be inferred thepresence of the pathogen 1520.

A variation on the process of FIG. 13 is possible as depicted in FIG.14.

In STEP 1 of FIG. 14, the pathogen 1520 in the form of mucus and/orsputum is deposited onto inside walls of the sample tube 70, 200.

In STEP 2, the second antibodies 1530 and their associated fluorophores1540 in the form of latex spheres are then released from one or more ofthe reservoirs 910, 920 in the sealing cap 900. The second antibodies1530 bind to the pathogen 1520 within the sample tube 200. The plunger230 is then used to mechanically concentrate the pathogens 1520 bound totheir second antibodies 1530 and associated relatively large latexspheres. Such an order of steps means that there is a relatively largefluid mass to collect than in FIG. 13; this relatively larger mass is ofbenefit where relatively little mucus and/or sputum is deposited withinthe tube 200.

In STEP 3, the pathogens 1520 bound to the second antibodies 1530 andtheir latex sphere laden fluorophores 1540 are then presented to theoptical aperture 450 whereat they bind to the first antibodies 1500immobilized onto the aperture 450. The pathogens 1520, the antibodies1500, 1530 and the fluorophore-laden latex spheres thereby become boundto the aperture 450 and fluorescence when interrogated with evanescentradiation to signal presence of the pathogen 1520 in the sample.

4.6.2 Competitive Binding Assay

The competitive binding assay is depicted in FIG. 15.

In STEP 1, the optical aperture 450 has bound thereto during fabricationthe first antibodies 1500. Moreover, analogues 1600 of the pathogen 1520to be detected are added to the aperture 450 for weakly binding to theimmobilized first antibodies 1500. The analogues 1600 have tightlyassociated thereto third antibodies 1610 bound to the fluorophores 1540;the fluorophores 1540 can, if required, be fluorophores bound in theaforesaid latex spheres.

In operation, the optical aperture 450 is interrogated using evanescentradiation to obtain a first fluorescence measurement. Next, a mucusand/or sputum sample is collected in the interior surface of the tube200. The sample is then mechanically concentrated using the plunger 230as described in the foregoing and finally deposited onto the opticalaperture 450.

In STEP 2, the pathogens 1520 in the mechanically-concentrated samplehave greater affinity for the first antibodies 1500 and competitivelydisplace the analogues 1600 which become detached and migrate with theirassociated fluorophores to regions remote from where the evanescentradiation propagates at the optical aperture 450. The optical aperture450 is then interrogated for a second time with evanescent waveradiation of identical amplitude to that use to obtain the firstmeasurement; a second fluorescence measurement is thereby obtained. Adifference between the first and second measurements is indicative ofthe number of displaced fluorophores and hence, by inference, thepresence of the pathogen 1520 in the collected sample.

4.6.3 Assay Detection Methods Not Involving Fluorescence or EvanescentWaves

The measurement system 10 can be adapted to utilize a detection andlabeling scheme that does not rely on evanescent wave excitation offluorescence. An example of such a scheme will be outlined:

Standard Method: STEP 1: Incubate sample with surface-bound IgG; anyanalyte present is immobilised at surface by IgG STEP 2: Rinse STEP 3:Incubate with labeled IgG. If any immobilised analyte is present,labeled IgG is immobilised at surface STEP 4: Rinse to remove unboundIgG and label STEP 5: Add developing agent if required STEP 6: Measureresult

With all such schemes, if monoclonal antibodies targeted at twodifferent epitopes are used, it is potentially possible to perform thetwo incubation steps simultaneously, thereby circumventing the need torinse between the at STEPS 2 and 4.

Possible labels include, but are not limited to, substances listed inTable 3.

TABLE 3 Chemical labels Technique Label Developing agent MeasurementChromogenic chromogenic Enzyme substrate Bulk colourimetry ELISA enzymeFluorogenic ELISA fluorogenic enzyme Enzyme substrate Bulk fluorescenceChemiluminescent chemiluminescent Chemiluminescent Chemiluminescenceassay molecule substrate at surface Radio immuno- Radio isotope NoneRadiation at surface assay (RIA) Colloidal gold Colloidal gold(optional) plating Colourimetry at surface assay solution to increasesize of colloids5.0 Applications for Use of the Measurement System

The biological measurement system 10 can be used in applications wherethe sample is not sputum and/or mucus. Possible other samples foranalysis by the system 10 include, for example, one or more of:

-   (a) blood;-   (b) urine;-   (c) pathogenic sera;-   (d) semen;-   (e) saliva;-   (f) tears; and-   (g) sweat.

Moreover, the measurement system 10 can also be adapted to interrogateairborne particles such as airborne micro-organisms, spores, pollen, orairborne dust (for example from chemical processing plants wheredangerous chemicals are used and/or manufactured).

The measurement system 10 described in the foregoing can be adapted foruse in the detection of many other bacterial and viral infectionsincluding, but not limited to:

-   (a) other forms of pneumonia such as influenzal pneumonia or viral    pneumonia;-   (b) tuberculosis;-   (c) malaria;-   (d) diptheria;-   (e) lupuserethemytosis;-   (f) pertussis;-   (g) other zymotic diseases;-   (h) streptococcus; and-   (i) staphylococcus.

The measurement system 10 can also be applied to detect viral particles,allergens or spores, pollen or other particles of a biological nature,or particles which are non-organic but can be detected by antibodies,nucleic acids or other suitable recognition groups. Such non-organicparticles can include airborne particles of toxic compounds, controllednarcotics, explosives or any other particles that are present in air,water and other liquids.

Moreover, the measurement system 10 can be adapted to detect sympatheticparticles such as indicators of certain forms of cancer.

Moreover, the measurement system 10 can be applied to detect particlesthat do not cause disease, such as antibodies. Thus, the measurementsystem 10 can be readily adapted for use in the early detection of HIVand AIDS, thereby being potentially valuable technology for countriessuch as South Africa which is having to cope with such diseases.

It will be appreciated that the aforementioned biological measurementsystem 10 can be modified. For example, although antibodies are used torecognise and bind particles to be interrogated for pathogens, otherrecognition groups can be employed. For example, one or more of thefollowing substances can be used:

-   (a) proteins such as enzymes;-   (b) aptamers of other sequences of nucleic acid or nucleic acid    analogues;-   (c) analogues of proteins;-   (d) artificial polypeptides; and-   (e) entire organisms.

If the particles in the sample are themselves fluorescent, they can beinterrogated directly to generate the radiation; such particlescircumvent the need for treatment with fluorescently-labeled antibodiesas described in the foregoing.

1. A biological measurement system for measuring the concentration ofcomponents included in a sample obtained in aerosol form from a patient,the system comprising: (a) collecting means for collecting the sample inaerosol form from a patient comprising a collection vessel with aninside surface, said vessel having an aperture for allowing the sampleto enter said vessel in aerosol form in order to be collected withinsaid vessel and deposited onto said inside surface; (b) concentratingmeans for mechanically collecting the sample from the inside surface ofthe collecting means, which when actuated, mechanically collects saidsample from said inside surface of said collecting means to form aspatially concentrated sample which is transferred to a region withinsaid vessel where the concentrated sample is optically interrogated,said concentrating means including means for scraping said insidesurface where the sample is deposited; (c) marking means for opticallylabeling the components present in the concentrated sample to producelabeled components; and (d) interrogating means for opticallyinterrogating said labeled components and thereby generating a measureof the concentration of components present in the sample.
 2. A systemaccording to claim 1, wherein said means for scraping is elasticallydeformable for spreading the spatially concentrated sample over anoptical interrogation region whereat the concentrated sample issubjected to optical interrogation.
 3. A system according to claim 1,wherein the marking means comprises at least one of a selective bindingassay and a competitive displacement assay for optically marking thepresence of the components by way of fluorescent markers, wherein saidfluorescent markers are bound to antibodies for use in at least one ofthe selective assay and the competitive assay; wherein said fluorescentmarkers comprise fluorophores bound to the antibodies by way of anintermediate carrier such that a plurality of fluorophores areassociated with each antibody, and wherein the intermediate carriercomprises latex spheres.
 4. A system according to claim 1, wherein saidinterrogating means comprises an optical evanescent detector fordetecting changes in optical responses induced by the presence of thecomponents.
 5. A system according to claim 4, wherein said evanescentdetector comprises: (a) at least one member selected from the groupconsisting of a diode laser and a LED as a source of interrogatingradiation for interrogating the concentrated sample; and (b) at leastone member selected from the group consisting of an avalanchephotodiode, a photodiode array and a photomultiplier tube as an opticaldetector for detecting fluorescent radiation emitted from theconcentrated sample in response to optical interrogation of the sample,said optical detector including means for generating a detection signalindicative of changes in fluorescence from the sample resulting from thepresence of the components in the sample.
 6. A system according to claim1, wherein said vessel is arranged to enclose the sample, therebypreventing personal contact with the sample when the system is in use.7. A system according to claim 6, wherein said collecting means is asingle-use disposable part.
 8. A system according to claim 1, whereinsaid vessel comprises vortex enhancing means for deposition of thesample within said vessel.
 9. A system according to claim 1, whereinsaid vessel comprising a filtering means for at least partiallyinhibiting spread of the components of the sample from said vessel. 10.A system according to claim 1, wherein the marking means includes lysingmeans for causing lysis of the components present in the sample, therebyenhancing measurement sensitivity of the system by increasing the numberof available potential optical labeling sites.
 11. A method of detectingat least one pathogen in at least one sample of the sputum/mucus from asubject comprising the steps of: (a) providing a biological measurementsystem comprising collecting means for collecting the sample in aerosolform from a patient comprising a collection vessel with an insidesurface, said vessel having an aperture for allowing the sample to entersaid vessel in aerosol form in order to be collected within said vesseland deposited onto said inside surface; concentrating means formechanically collecting the sample from the inside surface of thecollecting means, which when actuated, mechanically collects said samplefrom said inside surface of the said collecting means to form aspatially concentrated sample which is transferred to a region withinsaid vessel where the concentrated sample is optically interrogated,said concentrating means including means for scraping said insidesurface where the sample is deposited; marking means for opticallylabeling the components present in the concentrated sample to producelabeled components; and interrogating means for optically interrogatingsaid labeled components and thereby generating a measure of theconcentration of components present in the sample; (b) collecting saidone or more samples in aerosol form in the collecting means; (c)spatially concentrating the one or more samples in the concentratingmeans by mechanically collecting the sample from the inside surface ofthe collecting means; (d) optically labeling one or more pathogenspresent in said one or more samples; (e) optically interrogating thepathogens to achieve an optical response; and (f) determining from theoptical response of said one or more samples whether or not said one ormore pathogens are present in said one or more samples.
 12. A methodaccording to claim 11, wherein said steps (b), (c) and (d) of the methodis performed using evanescent-wave spectroscopy.
 13. A method accordingto claim 11 adapted for the detection of bacteria associated withpulmonary and pulmonary-related infections.
 14. A method according toclaim 11, wherein the inhalation of at least one of: esters, eatervapor, saline vapor, expectorant and menthol is used to assist releaseof bacteria-containing mucus from the trachea or form the upper lung ofa subject being tested.
 15. A method according to claim 11, wherein apartial negative pressure is employed to assist in obtaining said atleast one sample in aerosol form.
 16. A method according to claim 11,wherein said at least one sample comprises an aerosol of blood.
 17. Amethod according to claim 11, wherein analysis of said one or moresamples is performed using at least one of: (a) an ELISA chromogenicreaction; and (b) a surface acoustic wave (SAW) biosensor to detect anantigen in said one or more samples.
 18. A method according to claim 11,wherein said at least one sample comprises a bodily fluid in liquidform.
 19. A method according to claim 11, wherein said at least onesample comprises a bodily fluid in aerosol form.